Sclerotinia-resistant Brassica

ABSTRACT

The invention provides  Brassica  plants and lines having an improved  Sclerotinia sclerotiorum  Disease Incidence (SSDI %) score and represented by, or descended from, ATCC accession number PTA-6776 or PTA-6780.

REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No. 11/422,623filed Jun. 7, 2006, which claims priority to U.S. provisionalapplication 60/688,687 filed Jun. 9, 2005, both of which are herebyincorporated by reference.

TECHNICAL FIELD

The present invention relates to Sclerotinia resistant Brassica.

BACKGROUND OF THE INVENTION

Sclerotinia infects over 100 species of plants, including numerouseconomically important crops such as Brassica species, sunflowers, drybeans, soybeans, field peas, lentils, lettuce, and potatoes (Boland andHall, 1994). Sclerotinia sclerotiorum is responsible for over 99% ofSclerotinia disease, while Sclerotinia minor produces less than 1% ofthe disease. Sclerotinia produces sclerotia, irregularly-shaped, darkoverwintering bodies, which can endure in soil for four to five years.The sclerotia can germinate carpogenically or myceliogenically,depending on the environmental conditions and crop canopies. The twotypes of germination cause two distinct types of diseases. Sclerotiathat germinate carpogenically produce apothecia and ascospores thatinfect above-ground tissues, resulting in stem blight, stalk rot, headrot, pod rot, white mold, and blossom blight of plants. Sclerotia thatgerminate myceliogenically produce mycelia that infect root tissues,causing crown rot, root rot and basal stalk rot.

Sclerotinia causes Sclerotinia stem rot, also known as white mold, inBrassica, including canola. Canola is a type of Brassica having a lowlevel of glucosinolates and erucic acid in the seed. The sclerotiagerminate carpogenically in the summer, producing apothecia. Theapothecia release wind-borne ascospores that travel up to one kilometer.The disease is favoured by moist soil conditions (at least 10 days at ornear field capacity) and temperatures of 15-25° C., prior to and duringcanola flowering. The spores cannot infect leaves and stems directly;they must first land on flowers, fallen petals, and pollen on the stemsand leaves. Petal age affects the efficiency of infection, with olderpetals more likely to result in infection (Heran, et al., 1999). Thefungal spores use the flower parts as a food source as they germinateand infect the plant.

The severity of Sclerotinia in Brassica is variable, and is dependent onthe time of infection and climatic conditions (Heran, et al., 1999). Thedisease is favored by cool temperatures and prolonged periods ofprecipitation. Temperatures between 20 and 25° C. and relativehumidities of greater than 80% are required for optimal plant infection(Heran, et al., 1999). Losses ranging from 5 to 100% have been reportedfor individual fields (Manitoba Agriculture, Food and Rural Initiatives,2004). On average, yield losses equal 0.4 to 0.5 times the percentageinfection. For example, if a field has 20% infection (20/100 infectedplants), then the yield loss would be about 10%. Further, Sclerotiniacan cause heavy losses in wet swaths. Sclerotinia sclerotiorum causedeconomic losses to canola growers in Minnesota and North Dakota of 17.3,20.8 and 16.8 million dollars in 1999, 2000, and 2001, respectively.(Bradley, et al. 2006)

The symptoms of Sclerotinia infection usually develop several weeksafter flowering begins. The plants develop pale-grey to white lesions,at or above the soil line and on upper branches and pods. The infectionsoften develop where the leaf and the stem join because the infectedpetals lodge there. Once plants are infected, the mold continues to growinto the stem and invade healthy tissue. Infected stems appear bleachedand tend to shred. Hard black fungal sclerotia develop within theinfected stems, branches, or pods. Plants infected at flowering producelittle or no seed. Plants with girdled stems wilt and ripen prematurely.Severely infected crops frequently lodge, shatter at swathing, and makeswathing more time consuming. Infections can occur in all above-groundplant parts, especially in dense or lodged stands, where plant-to-plantcontact facilitates the spread of infection. New sclerotia carry thedisease over to the next season.

Conventional methods for control of Sclerotinia diseases include (a)chemical control, (b) disease resistance and (c) cultural control, eachof which is described below.

(a) Fungicides such as benomyl, vinclozolin and iprodione remain themain method of control of Sclerotinia disease (Morall, et al., 1985; Tu,1983). Recently, additional fungicidal formulations have been developedfor use against Sclerotinia, including azoxystrobin, prothioconazole,and boscalid. (Johnson, 2005) However, use of fungicide is expensive andcan be harmful to the user and environment. Further, resistance to somefungicides has occurred due to repeated use.

(b) In certain cultivars of bean, safflower, sunflower and soybean, someprogress has been made in developing partial (incomplete) resistance.Partial resistance is often referred to as tolerance. However, successin developing partial resistance has been very limited, probably becausepartial physiological resistance is a multigene trait as demonstrated inbean (Fuller, et al., 1984). In addition to partial physiologicalresistance, some progress has been made to breed for morphologicaltraits to avoid Sclerotinia infection, such as upright growth habit,lodging resistance and narrow canopy. For example, bean plants withpartial physiological resistance and with an upright stature, narrowcanopy and indeterminate growth habit were best able to avoidSclerotinia (Saindon, et al., 1993). Early maturing cultivars ofsafflower showed good field resistance to Sclerotinia. Finally, insoybean, cultivar characteristics such as height, early maturity andgreat lodging resistance result in less disease, primarily because of areduction of favorable microclimate conditions for the disease. (Bolandand Hall, 1987; Buzzell, et al., 1993)

(c) Cultural practices such as using pathogen-free or fungicide-treatedseed, increasing row spacing, decreasing seeding rate to reducesecondary spread of the disease, and burying sclerotia to preventcarpogenic germination may reduce Sclerotinia disease but noteffectively control the disease.

All Canadian canola genotypes are susceptible to Sclerotinia stem rot(Manitoba Agriculture, Food and Rural Initiatives, 2004). This includesall known spring petalled genotypes of canola quality. There is also noresistance to Sclerotinia in Australian canola varieties.(Hind-Lanoiselet, et al., 2004) Some varieties with certainmorphological traits are better able to withstand Sclerotinia infection.For example, Polish varieties (Brassica rapa) have lighter canopies andseem to have much lower infection levels. In addition, petal-lessvarieties (apetalous varieties) avoid Sclerotinia infection to a greaterextent (Okuyama, et al., 1995; Fu, 1990). Other examples ofmorphological traits which confer a degree of reduced fieldsusceptibility in Brassica genotypes include increased standability,reduced petal retention, branching (less compact and/or higher), andearly leaf abscission. Jurke and Fernando, (2003) screened eleven canolagenotypes for Sclerotinia disease incidence. Significant variation indisease incidence was explained by plant morphology, and the differencein petal retention was identified as the most important factor. However,these morphological traits alone do not confer resistance toSclerotinia, and all canola products in Canada are consideredsusceptible to Sclerotinia.

Winter canola genotypes are also susceptible to Sclerotinia. In Germany,for example, no Sclerotinia-resistant varieties are available. (Specht,2005) The widely-grown German variety Express is considered susceptibleto moderately susceptible and belongs to the group of less susceptiblevarieties/hybrids. (See, Table 4)

Spraying with fungicide is the only means of controlling Sclerotinia incanola crops grown under disease-favorable conditions at flowering.Typical fungicides used for controlling Sclerotinia on Brassica includeRovral™ from Bayer and Ronilan™/Lance™ from BASF. The active ingredientin Lance™ is Boscalid, and it is marketed as Endura™ in the UnitedStates. The fungicide should be applied before symptoms of stem rot arevisible and usually at the 20-30% bloom stage of the crop. If infectionis already evident, there is no use in applying fungicide as it is toolate to have an effect. Accordingly, growers must assess their fieldsfor disease risk to decide whether to apply a fungicide. This can bedone by using a government provided checklist or by using a petaltesting kit. Either method is cumbersome and prone to errors.(Hind-Lanoiselet, 2004; Johnson, 2005)

Numerous efforts have been made to develop Sclerotinia resistantBrassica plants. Built-in resistance would be more convenient,economical, and environmentally-friendly than controlling Sclerotinia byapplication of fungicides. Since the trait is polygenic it would bestable and not prone to loss of efficacy, as fungicides may be.

Spring canola (Brassica napus subsp. oleifera var. annua) differs fromwinter canola (Brassica napus subsp. oleifera var. biennis) primarily inthe absence of an obligate vernalization requirement. Asiatic rapeseed,and canola versions, have a low to intermediate requirement forvernalization, and are known as semi-winter types. While winter canolacannot finish its reproduction cycle when planted in the spring, earlyspring planting and exposure to cold enables Asiatic material to flowerand set seed. Asiatic material cannot finish its reproduction cycle ifplanted in late spring. In controlled conditions, winter materialrequires 12-14 weeks of vernalization while Asiatic material requires2-8 weeks. Table 1 summarizes the differences between winter,semi-winter (Asiatic) and spring canola varieties.

TABLE 1 Main determinations of growth habit in Brassica napus materialsSemi Winter Type Spring* Spring (Asiatic) Winter Growing Canada,Australia China, Europe areas Europe Japan Vernalization None None 2-8weeks 12-14 weeks Requirement intermediate strong or full Time of SpringFall Fall Fall seeding (Increasing (Decreasing (Decreasing (DecreasingDay Length) Day Length) Day Length) Day Length) Number of 30-90 90-150120-180 150-270 days until flowering *Canadian, European and Australianspring materials can be planted and grown in any environment or seedingtime for spring canola.

Some Chinese (semi-winter) cultivars of rapeseed/canola are partiallyresistant to Sclerotinia. For example, ChunYun, et al., 2003; HanZhong,et al., 2004; XeiXin, et al., 2002; YongJu et al., 2000; ChaoCai, etal., 1998 describe partially resistant varieties of rapeseed. However,some of these varieties are not canola quality, and all of them requirevernalization. The partial field resistance in Chinese varietiesoriginated mostly from the rapeseed variety Zhong you 821. Despiteimprovements in partial resistance in Zhong you 821, its reaction todisease is less stable under environmental conditions favorable fordevelopment of Sclerotinia (Yunchang, et al., 1999). This indicates alower level of partial resistance (Li, et al., 1999).

Some Japanese cultivars of rapeseed have partial stem resistance toSclerotinia. Partial stem resistance was detected by indoor tests incomparison with winter canola (Brun, et al., 1987). However, thesevarieties are not canola quality and are semi-winter types (see, Table1).

Breeding for Sclerotinia field resistance in canola has been verydifficult due to the quantitative nature of this trait. Further, theincorporation of physiological resistance with morphological traits thatavoid or reduce infection multiplies the complexity of breeding forresistance. In addition, it has been very difficult to screen forresistance because of the direct environment interaction (i.e.,temperature and humidity requirements, as well as microenvironmentrequirements) with the plant population. As stated above, there are noCanadian spring Brassica varieties with resistance to Sclerotinia, thisdespite many years of co-evolution and environmental pressure to selectfor this trait. The highest available level of field resistance inrapeseed (and recently some canola materials) was attained via breedingefforts in China as described with Zhong you 821 (Yunchang, et al.,1999). The levels of such partial resistance or tolerance are relativelylow as fungicide applications are still recommended on all rapeseed andcanola materials in China (verbal communication) (Baocheng, et al.,1999). Clearly, Brassica and canola varieties with high levels ofresistance to Sclerotinia are not found in nature.

Canola quality Brassica napus was developed in the 1970's. Despite 30years of extensive breeding efforts, no canola varieties resistant toSclerotinia have previously been developed. The breeding effortsincluded quantitative trait loci analysis (Zhao-Jianwei, et al., 2003),mutagenesis breeding (Mullins, et al., 1999; Wu-Yanyou, et al., 1996;LiangHong, et al., 2003), extensive screening efforts (Sedune, et al.,1989; Zhao, et al., 2004); and screening for expressed sequence tags(ESTs) (Rugang, et al., 2004), to name a few. Several spring canolavarieties with moderate tolerance to Sclerotinia have been developed(Ahmadi, et al., 2000a; Ahmadi, et al., 2000b; BaoMing, et al., 1999;and Liu, et al., 1991), however the level of tolerance is low and thelines cannot withstand high disease pressure. Recently, transgeniccanola has been developed carrying an oxalic oxidase gene (U.S. Pat. No.6,166,291 and divisional patents thereof); however there are regulatoryand social issues associated with transgenic plants. Winter canolagenotypes with resistance to Sclerotinia are also needed as indicated byfungicide applications (Johnson, 2005). Accordingly, significanttechnical human intervention is required to breed canola varieties thatare resistant to Sclerotinia.

SUMMARY OF THE INVENTION

An aspect of the present invention is to provide a Brassica plant orgroup of plants with improved resistance to Sclerotinia sclerotiorum. Inone aspect, the invention provides a spring Brassica napus plant orgroup of plants, the plant or group of plants being representative of apopulation wherein the population has an average Sclerotiniasclerotiorum Disease Incidence (SSDI %) score which is less than about60% of the SSDI % score of Pioneer Hi-Bred variety 46A76, or of PioneerHi-Bred variety 46A65, or of the mean SSDI % score of the two varieties,under the same environmental and disease conditions in the field. TheBrassica napus plant or group of plants may also be representative of apopulation wherein the population has an average Sclerotiniasclerotiorum Disease Incidence (SSDI %) score which is less than 50%,35% or 20% of the score of Pioneer Hi-Bred variety 46A76 or of PioneerHi-Bred variety 46A65 or of the mean score of the two varieties.

Another aspect of the present invention is to provide a winter Brassicanapus plant or group of plants, the plant or group of plants beingrepresentative of a population wherein the population has an averageSclerotinia sclerotiorum Disease Incidence (SSDI %) score which is lessthan about 60% of the SSDI % score of the variety Columbus, or of thevariety Express, or of the mean SSDI % score of the two varieties, underthe same environmental and disease conditions in the field. The winterBrassica napus plant or group of plants may also be representative of apopulation wherein the population has an average Sclerotiniasclerotiorum Disease Incidence (SSDI %) score which is less than 50%,35% or 20% of the score of the variety Columbus or of the varietyExpress or of the mean score of the two varieties.

Another aspect of the present invention is to provide a spring Brassicanapus plant or group of plants, the plant or group of plantsrepresenting a population characterized by at least the followingtraits: (a) a solid component of the seed of the population comprising aglucosinolate level of less than 30 μmoles per gram of oil-free solid,(b) oil of the seed of the plant comprising less than 2% erucic acid,(c) a 50% flowering time of between about 30 to 90 days, and (d) an SSDI% score which is less than about 60% of the SSDI % score of PioneerHi-Bred variety 46A76, or of Pioneer Hi-Bred variety 46A65, or of themean score of the two varieties, under the same environmental anddisease conditions in the field. The Brassica napus plant may also berepresentative of a population wherein the population has an averageSclerotinia sclerotiorum Disease Incidence (SSDI %) score which is lessthan 50%, 35% or 20% of the score of Pioneer Hi-Bred variety 46A76 or ofPioneer Hi-Bred variety 46A65 or of the mean score of the two varieties.

Another aspect of the present invention is to provide a winter Brassicanapus plant or group of plants, the plant or group of plantsrepresenting a population characterized by at least the followingtraits: (a) a solid component of the seed of the population comprising aglucosinolate level of less than 30 μmoles per gram of oil-free solid,(b) oil of the seed of the population comprising less than 2% erucicacid, (c) a 50% flowering time of between about 30 to 90 days, and (d)an SSDI % score which is less than about 60% of the SSDI % score of thevariety Columbus, or of the variety Express, or of the mean SSDI % scoreof the two varieties, under the same environmental and diseaseconditions in the field. The winter Brassica napus plant or group ofplants may also be representative of a population wherein the populationhas an average Sclerotinia sclerotiorum Disease Incidence (SSDI %) scorewhich is less than 50%, 35% or 20% of the score of the variety Columbusor of the variety Express or of the mean score of the two varieties.

The Brassica napus plant may represent a spring Brassica napus line asfollows:

-   -   (a) an S3 bulk increase of 03SN40341, deposited under ATCC        accession no. PTA-6776; or a doubled-haploid line derived from        03SN40341 and deposited under ATCC accession no. PTA-6780.    -   (b) an S3 bulk increase of 03SN40441, deposited under ATCC        accession no. PTA-6779; or a doubled-haploid line derived from        03SN40441 and deposited under ATCC accession no. PTA-6778.    -   (c) an F4 bulk increase of 02SN41269, deposited under ATCC        accession no. PTA-6777; or a doubled-haploid line derived from        02SN41269 and deposited under ATCC accession no. PTA-6781.    -   (d) An S2 bulk designated 04SN41433, deposited under NCIMB        accession no. 41389 or a doubled-haploid line derived from        04SN41433 and deposited under NCIMB accession no. 41391.    -   (e) an S2 bulk designated 04SN41415, deposited under NCIMB        accession no. 41388, or a doubled-haploid line derived from        04SN41415 and deposited under NCIMB accession no. 41390.

(See also, Table 11a.)

The Brassica napus plant may represent a winter Brassica napus line asfollows:

-   -   (a) an F4 bulk increase of line 04CWB930128, deposited under        NCIMB accession no. 41396.    -   (b) an F4 bulk increase of line 04CWB930127, deposited under        NCIMB accession no. 41395.    -   (c) an F4 bulk increase of line 04CWB930081, deposited under        NCIMB accession no. 41393.    -   (d) an F4 bulk increase of line 04CWB930111, deposited under        NCIMB accession no. 41394.    -   (e) an F4 bulk increase of line 04CWB930144, deposited under        NCIMB accession no. 41398    -   (f) an F4 bulk increase of line 04CWB930015, deposited under        NCIMB accession no. 41392.    -   (g) an F4 bulk increase of line 04CWB930135, deposited under        NCIMB accession no. 41397.

(See also, Table 11b.)

Another aspect of the present invention is to provide a descendent plantof any of the Brassica plants of the above-mentioned aspects, whereinthe descendent plant is characterized by at least the following traits:(a) a solid component of the seed of the plant comprising aglucosinolate level of less than 30 μmoles per gram of oil-free solid,(b) oil of the seed of the plant comprising less than 2% erucic acid,(c) a 50% flowering time of between about 30 to 90 days, and (d) beingrepresentative of a population having an SSDI % score which is less thanabout 60% of the SSDI % score (1) of Pioneer Hi-Bred variety 46A76, orof Pioneer Hi-Bred variety 46A65, or of the mean score of the twovarieties, where the descendent plant has a spring growth habit, or (2)of the SSDI % score of the variety Columbus, or of the variety Express,or of the mean SSDI % score of the two varieties, where the descendentplant has a winter growth habit, under the same environmental anddisease conditions in the field. The descendent Brassica napus plant mayalso represent a population having an average level of Sclerotiniaincidence of less than 50%, 35% or 20% of the SSDI % score of (1)Pioneer Hi-Bred variety 46A76 or Pioneer Hi-Bred variety 46A65 or themean score of the two varieties, where the descendent plant has a springgrowth habit, or (2) the variety Columbus or the variety Express or themean score of the two varieties, where the descendent plant has a wintergrowth habit, under the same environmental and disease conditions in thefield.

Another aspect of the invention is to provide a Brassica napus plant orgroup of plants, the plant or group of plants having physiologicaltraits, or a combination of morphological and physiological traitsfunctioning in synchrony, to reduce disease development, wherein theplant or group of plants is representative of a population, saidpopulation having an SSDI % score which is less than about 60% of theSSDI % score of (1) Pioneer Hi-Bred variety 46A76, or Pioneer Hi-Bredvariety 46A65, or the mean score of the two varieties, where thepopulation has a spring growth habit, or (2) the variety Columbus or thevariety Express or the mean score of the two varieties, where thepopulation has a winter growth habit, under the same environmental anddisease conditions in the field.

Another aspect of the invention is to provide progeny of any of theBrassica napus plants discussed above, said progeny produced byextracting the Sclerotinia resistant trait by doubled haploidy, andwherein a homogeneous population comprising said progeny has an SSDI %score which is less than about 60% of the SSDI % score of (1) PioneerHi-Bred variety 46A76 or Pioneer Hi-Bred variety 46A65 or the mean scoreof the two varieties, where the population has a spring growth habit, or(2) the variety Columbus or the variety Express or the mean score of thetwo varieties, where the population has a winter growth habit, under thesame environmental and disease conditions in the field.

Further, the invention also provides a doubled haploid line producedfrom any of the Brassica napus plants discussed above, seed from any ofthe plants, crushed Brassica napus seed from any of the plants, plantcells from any of the plants, and cellular plant material from any ofthe plants, for example, pollen or ovule material.

Another aspect of the invention is to provide a method for screening forresistance of a plant to Sclerotinia under controlled environmentalconditions, comprising, (a) inoculating the plant growing in controlledenvironmental conditions with a low-nutrient PDA plug comprisingmycelium of Sclerotinia, and (b) screening for resistance of the plantto Sclerotinia. The plug may be attached to the plant by anentomological needle. The plug may be about 3 mm. The controlledenvironmental conditions may comprise controlled humidity.

Another aspect of the invention is to provide a method for screening aplant growing in the field for resistance to Sclerotinia, comprising,(a) inoculating the plant with Sclerotinia, (b) irrigating the plantwith water, wherein the water is low in, or free of, ions which couldbind with oxalic acid; (c) maintaining a pre-determined threshold ofcontinuous wetness on the plant, and (d) screening for resistance of theplant to Sclerotinia. Inoculation may be accomplished using a carriermaterial. The carrier may be seed, such as Niger seed, colonized withSclerotinia, and may be disseminated at a rate of about 5-20 kg/ha. Thewater may be deionized water, distilled water, runoff water or collectedrainwater. The method may further comprise use of a netting enclosure toprovide a controlled microenvironment.

Another aspect of the invention is to provide a method of producing asuccessive generation of a Brassica napus line 03SN40341 having an SSDI% score which is less than about 60% of the SSDI % score of PioneerHi-Bred variety 46A76, or of Pioneer Hi-Bred variety 46A65, or of themean score of the two varieties, under the same environmental anddisease conditions in the field, comprising, (a) crossing Brassica napusline 03SN40341 with itself or with another Brassica plant to yield aBrassica line 03SN40341-derived progeny Brassica seed, (b) growing theBrassica napus seed of step (a) to yield an additional Brassica line03SN40341-derived Brassica plant, (c) optionally repeating the crossingand growing of steps (a) and (b) for successive generations to producefurther plants derived from Brassica napus line 03SN40341, and (d)selecting a descendent plant wherein a said plant represents apopulation of plants having an SSDI % score which is less than about 60%of the SSDI % score of Pioneer Hi-Bred variety 46A76, or of PioneerHi-Bred variety 46A65, or of the mean score of the two varieties, underthe same environmental and disease conditions in the field. Theinvention also provides similar methods for lines 03SN40441, 02SN41269,04DHS12921, 04DHS11319, 04DHS11418, 04SN41433, 04SN41415, 05DHS12897,and 04DHS12879. A population represented by the descendent plant mayhave an SSDI % score which is less than about 50%, 35%, or 20% of theSSDI % score of Pioneer Hi-Bred variety 46A76, or of Pioneer Hi-Bredvariety 46A65, or of the mean score of the two varieties, under the sameenvironmental and disease conditions in the field.

Another aspect of the invention is to provide a method of producing asuccessive generation of a Brassica napus line 04CWB930127 having anSSDI % score which is less than about 60% of the SSDI % score of thevariety Columbus, or of the variety Express, or of the mean score of thetwo varieties, under the same environmental and disease conditions inthe field, comprising, (a) crossing Brassica napus line 04CWB930127 withitself or with another Brassica plant to yield a Brassica line04CWB930127-derived progeny Brassica seed, (b) growing the Brassicanapus seed of step (a) to yield an additional Brassica line04CWB930127-derived Brassica plant, (c) optionally repeating thecrossing and growing of steps (a) and (b) for successive generations toproduce further plants derived from Brassica napus line 04CWB930127, and(d) selecting a descendent plant wherein said plant represents apopulation of plants having an SSDI % score which is less than about 60%of the SSDI % score of the variety Columbus or the variety Express orthe mean score of the two varieties, under the same environmental anddisease conditions in the field. The invention also provides similarmethods for lines 04CWB930128, 04CWB930081, 04CWB930111, 04CWB930144,04CWB930135, and 04CWB930015. A population represented by the descendentplant may have an SSDI % score which is less than about 50%, 35%, or 20%of the SSDI % score of the variety Columbus or the variety Express orthe mean of the two varieties, under the same environmental and diseaseconditions in the field.

Another aspect of the present invention is to provide use of a springBrassica napus plant, the plant being representative of a populationwhich has an SSDI % score which is less than about 60% of the SSDI %score of Pioneer Hi-Bred variety 46A76, or of Pioneer Hi-Bred variety46A65, or of the mean score of the two varieties, under the sameenvironmental and disease conditions in the field, for growing a crop,for oil and meal production, or for breeding a new Brassica line. Theplant may be designated 03SN40341, 03SN40441, 02SN41269, 04DHS12921,04DHS11319, or 04DHS11418, representative seed being deposited underATCC accession no: PTA-6776, PTA-6779, PTA-6777, PTA-6781, PTA-6780, orPTA-6778, respectively, said seed deposited on or about Jun. 8, 2005; ormay be designated 04SN41433, 04SN41415, 05DHS12897, or 04DHS12879,representative seed being deposited under NCIMB accession no 41389,41388, 41391, or 41390, respectively.

Another aspect of the present invention is to provide use of a winterBrassica napus plant, the plant being representative of a populationwhich has an SSDI % score which is less than about 60% of the SSDI %score of the variety Columbus, or of the variety Express, or of the meanscore of the two varieties, under the same environmental and diseaseconditions in the field, for growing a crop, for oil and mealproduction, or for breeding a new Brassica line. The plant may bedesignated 04CWB930127, 04CWB930128, 04CWB930081, 04CWB930111,04CWB930144, 04CWB930135, or 04CWB930015, representative seed beingdeposited under NCIMB accession no: 41395, 41396, 41393, 41394, 41398,41397, or 41392, respectively.

Another aspect of the present invention is to provide a method ofproducing a canola oil, comprising (a) crushing seeds produced by aBrassica napus plant which may be designated 03SN40341, 03SN40441,02SN41269, 04DHS12921, 04DHS11319, or 04DHS11418, representative seedbeing deposited under ATCC accession no: PTA-6776, PTA-6779, PTA-6777,PTA-6781, PTA-6780, or PTA-6778, respectively, said seed deposited on orabout Jun. 8, 2005; or may be designated 04SN41433, 04SN41415,05DHS12897, or 04DHS12879, representative seed being deposited underNCIMB accession no. 41389, 41388, 41391, or 41390, respectively; or adescendent of any said plants, wherein the plant or descendent plant isrepresentative of a population having an SSDI % score which is less thanabout 60% of the SSDI % score of Pioneer Hi-Bred variety 46A76, or ofPioneer Hi-Bred variety 46A65, or of the mean score of the twovarieties, under the same environmental and disease conditions in thefield, (b) extracting a crude oil from said crushed seeds, andoptionally (c) refining, bleaching and deodorizing said crude oil toproduce the canola oil.

Another aspect of the present invention is to provide a method ofproducing a canola oil, comprising (a) crushing seeds produced by aBrassica napus plant which may be designated 04CWB930127, 04CWB930128,04CWB930081, 04CWB930111, 04CWB930144, 04CWB930135, or 04CWB930015,representative seed being deposited under NCIMB accession no: 41395,41396, 41393, 41394, 41398, 41397, or 41392, respectively, or may be adescendent of any said plants, wherein the plant or descendent plant isrepresentative of a population having an SSDI % score which is less thanabout 60% of the SSDI % score of the variety Columbus, or of the varietyExpress, or of the mean score of the two varieties, under the sameenvironmental and disease conditions in the field, (b) extracting acrude oil from said crushed seeds, and optionally (c) refining,bleaching and deodorizing said crude oil to produce the canola oil.Another aspect of the present invention is to provide a Brassica napusplant as discussed above, further having a level of blackleg(Leptosphaeria maculans) resistance greater than Pioneer Hi-Bred variety46A76 under the same environmental and disease conditions in the field.The plant may be designated 03SN40341, 03SN40441, 02SN41269, 04DHS12921,04DHS11319, or 04DHS11418, representative seed being deposited underATCC accession no: PTA-6776, PTA-6779, PTA-6777, PTA-6781, PTA-6780, orPTA-6778, respectively, said seed deposited on or about Jun. 8, 2005; ormay be designated 04SN41433, 04SN41415, 05DHS12897, or 04DHS12879,representative seed being deposited under NCIMB accession no 41389,41388, 41391, or 41390, respectively; or may be a descendent of any saidplants, wherein the plant or descendent plant is representative of apopulation having an SSDI % score which is less than about 60% of theSSDI % score of Pioneer Hi-Bred variety 46A76, or of Pioneer Hi-Bredvariety 46A65, or of the mean score of the two varieties, under the sameenvironmental and disease conditions in the field, and further has anaverage level of blackleg resistance greater than that of PioneerHi-Bred variety 46A76 under the same environmental and diseaseconditions in the field. Also provided is a plant cell from the plant.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a histogram of cycles 5 to 10 of Population T from 2000-2005under extreme disease pressure field research conditions. The Y-axisshows frequency of progenies. The X-axis shows the 1-9 SSDI Sclerotiniarating as described in Table 4.

FIG. 2 shows agronomic and Sclerotinia data for specificSclerotinia-resistant spring canola lines. Sclerotinia data areexpressed as % of 46A76, 46A65 and their mean. Part A includes agronomicdata and Sclerotinia data under extreme disease pressure field researchconditions. Part B includes natural field data. Part C shows combinedresults of Part A and Part B. Part D is Part B with data of one naturalfield trial (NDSU 2005) omitted. Part E shows combined results of Part Aand Part D.

DETAILED DESCRIPTION OF THE INVENTION I. Overview

The present invention discloses the first low erucic acid and lowglucosinolate canola lines having a spring or winter habit andexhibiting high levels of field resistance to Sclerotinia. Fieldresistance is based on an accumulation of conventional partialphysiological resistance to Sclerotinia in combination withmorphological traits that function in synchrony to reduce diseasedevelopment.

There are several aspects of this invention.

The first aspect is the development of Sclerotinia resistant canolalines. This aspect of the invention is described in examples 1, 2, 3, 4,8 and 9. This is the first report of spring canola lines having anaverage level of Sclerotinia incidence of less than about 60% of theincidence level of Pioneer Hi-Bred variety 46A76, or of Pioneer Hi-Bredvariety 46A65, or of the mean score of the two varieties under the sameenvironmental and disease conditions in the field, as measured by theSSDI % score; or of winter canola lines having an average level ofSclerotinia incidence of less than about 60% of the incidence level ofthe variety Columbus, or the variety Express, or of the mean score ofthe two varieties under the same environmental and disease conditions inthe field, as measured by the SSDI % score. The direct human technicalintervention to genetically manipulate and pyramid multiplephysiological and morphological traits during six years of breeding andselection efforts (2000-2005) stemming from 15 years of research (1991to 2005) has resulted in spring canola lines with resistance toSclerotinia. Seed deposits representing the improved lines have beenmade as detailed elsewhere herein, including Tables 11a and 11b.

The second aspect of the invention is developing canola lines with thecombination of Sclerotinia resistance and blackleg resistance. Thebreeding and selection efforts described in examples 1, 2, 3 and 4 notonly produced lines with Sclerotinia resistance, but also produced lineshaving blackleg resistance. The pyramiding of multiple physiological andmorphological traits during six years of breeding and selection stemmingfrom fifteen years of research resulted in lines with resistance toSclerotinia, and also resistance to blackleg. This aspect of theinvention is described in example 5.

The third aspect of the invention is the development of methodologies toscreen for Sclerotinia resistance in the greenhouse or growth room andin the field. Development of these methodologies was one of the criticalsuccess factors in developing the Sclerotinia-resistant lines andbackleg-resistant lines of the invention described in examples 1, 2, 3,4 and 5. This aspect is described in examples 6 and 7.

II. Canola Breeding Techniques

Canola breeding programs utilize techniques such as mass and recurrentselection, backcrossing, pedigree breeding and doubled haploiddevelopment. For a general description of rapeseed and canola breeding,see, R. K. Downey and G. F. W. Rakow, 1987: Rapeseed and Mustard (In:Fehr, W. R. (ed.), Principles of Cultivar Development, 437-486. NewYork: Macmillan and Co.); Thompson, K. F., 1983: Breeding winter oilseedrape Brassica napus. Advances in Applied Biology 7:1-104; and OilseedRape, Ward, et al., Farming Press Ltd., Wharefedale Road, Ipswich,Suffolk (1985), each of which is hereby incorporated by reference.

A cross between two different homozygous lines produces a uniformpopulation of hybrid plants (also called F1 hybrid plants) that may beheterozygous for many gene loci. A cross of two heterozygous plants thatdiffer at a number of gene loci will produce a population of plants thatdiffer genetically and will not be uniform. Regardless of parentage,plants that have been self-pollinated and selected for type for manygenerations become homozygous at almost all gene loci and produce auniform population of true pureline progeny. The term “inbred” as usedherein refers to a homozygous plant or a collection of homozygousplants. Those of ordinary skill will understand that some residualheterozygosity may exist in inbreds.

Choice of breeding or selection methods depends on the mode of plantreproduction, the heritability of the trait(s) being improved, and thetype of variety used commercially (e.g., F1 hybrid variety, purelinevariety, etc.). For highly heritable traits, a choice of superiorindividual plants evaluated at a single location will be effective,whereas for traits with low heritability, selection should be based onmean values obtained from replicated evaluations of families of relatedplants. Popular selection methods commonly include pedigree selection,modified pedigree selection, mass selection, and recurrent selection.

The complexity of inheritance influences choice of the breeding method.In general, breeding starts with the crossing of two genotypes, each ofwhich may have one or more desirable characteristics that is lacking inthe other or which complements the other. If the two original parents donot provide all the desired characteristics, other sources can beincluded by making more crosses. In each successive filial generation,F1 to F2; F2 to F3; F3 to F4; F4 to F5, etc., plants are selfed toincrease the homozygosity of the line. Typically in a breeding programfive or more generations of selection and selfing are practiced toobtain a homozygous plant.

Pedigree breeding is commonly used for the improvement ofself-pollinating crops. Two parents that possess favorable,complementary traits are crossed to produce an F1. An F2 population isproduced by selfing one or several F1's or by sib-pollinating two F1's.Selection of the best individuals may begin in the F2 population; then,beginning in the F3, the best individuals in the best families areselected. Replicated testing of families can begin in the F4 generationto improve the effectiveness of selection for traits with lowheritability. At an advanced stage of inbreeding (i.e., F6 and F7), thebest lines or mixtures of phenotypically similar lines are tested forpotential release as new varieties.

Backcross breeding has been used to transfer genes for simply inherited,highly heritable traits from a donor parent into a desirable, optimallyhomozygous, variety that is utilized as the recurrent parent. The sourceof the traits to be transferred is called the donor parent. After theinitial cross, individuals possessing the desired trait or traits of thedonor parent are selected and then repeatedly crossed (backcrossed) tothe recurrent parent. The resulting plant is expected to have theattributes of the recurrent parent plus the desirable trait or traitstransferred from the donor parent. This approach has been usedextensively for breeding disease-resistant varieties.

Each canola breeding program should include a periodic, objectiveevaluation of the efficiency of the breeding procedure. Evaluationcriteria vary depending on the goal and objectives, but should includegain from selection per year based on comparisons to an appropriatestandard, overall value of the advanced breeding lines, and number ofsuccessful varieties produced per unit of input (e.g., per year, perdollar expended, etc.).

Various recurrent selection techniques are used to improvequantitatively inherited traits controlled by numerous genes. The use ofrecurrent selection in self-pollinating crops depends on the ease ofpollination and the number of hybrid offspring from each successfulcross.

Mass selection and recurrent selection can be used to improvepopulations of either self- or cross-pollinated crops. A geneticallyvariable population of heterozygous individuals is either identified orcreated by intercrossing several different parents. The best plants areselected based on individual superiority, outstanding progeny, orexcellent combining ability. The selected plants are intercrossed toproduce a new population in which further cycles of selection arecontinued.

The single-seed descent procedure in the strict sense refers to plantinga segregating population, harvesting a sample of one seed per plant, andusing the one-seed sample to plant the next generation. When thepopulation has been advanced from the F2 to the desired level ofinbreeding, the plants from which lines are derived will each trace todifferent F2 individuals. The number of plants in a population declineseach generation due to the failure of some seeds to germinate or due tothe failure of some plants to produce at least one seed. As a result,not all of the F2 plants originally sampled in the population will berepresented by a progeny when generation advancement is completed.

In a multiple-seed procedure, canola breeders commonly harvest one ormore pods, or siliques, from each plant in a population and thresh themtogether to form a bulk. Part of the bulk is used to plant the nextgeneration and part is put in reserve. The procedure has been referredto as modified single-seed descent or the pod-bulk technique.

The multiple-seed procedure has been used to save labor at harvest. Itis considerably faster to thresh pods with a machine than to remove oneseed from each by hand for the single-seed procedure. The multiple-seedprocedure also makes it possible to plant the same number of seeds of apopulation each generation of inbreeding. Enough seeds are harvested tomake up for those plants that did not germinate or produce seed. Ifdesired, the doubled haploid method can be used to extract homogeneouslines, thereby increasing the supply of seed with a desired genotype.

Molecular markers including techniques such as Isozyme Electrophoresis,Restriction Fragment Length Polymorphisms (RFLPs), Randomly AmplifiedPolymorphic DNAs (RAPDs), Arbitrarily Primed Polymerase Chain Reaction(AP-PCR), DNA Amplification Fingerprinting (DAF), Sequence CharacterizedAmplified Regions (SCARs), Amplified Fragment Length Polymorphisms(AFLPs), Simple Sequence Repeats (SSRs) and Single NucleotidePolymorphisms (SNPS) may be used in plant breeding methods. One use ofmolecular markers is Quantitative Trait Loci (QTL) mapping. QTL mappingis the use of markers which are known to be closely linked to allelesthat have measurable effects on a quantitative trait. Selection in thebreeding process is based upon the accumulation of markers linked to thepositive effecting alleles and/or the elimination of the markers linkedto the negative effecting alleles from the plant's genome.

Molecular markers can also be used during the breeding process for theselection of qualitative traits. For example, markers closely linked toalleles, or markers containing sequences within the actual alleles ofinterest, can be used to select plants that contain the alleles ofinterest during a backcrossing breeding program. The markers can also beused to select for the genome of the recurrent parent and against themarkers of the donor parent. Using this procedure can minimize theamount of genome from the donor parent that remains in the selectedplants. It can also be used to reduce the number of crosses back to therecurrent parent needed in a backcrossing program. The use of molecularmarkers in the selection process is often called Genetic Marker EnhancedSelection or Marker Assisted Selection (MAS).

The production of doubled haploids (Swanson, et al., 1987) can also beused for the development of inbreds in the breeding program. After across is made, doubled haploid methods can be used to quickly obtain ahomozygous plant. In Brassica napus, microspore culture technique isused in producing haploid embryos. The haploid embryos are thenregenerated on appropriate media as haploid plantlets, doublingchromosomes of which results in doubled haploid plants. This can beadvantageous because the process omits the generations of selfing neededto obtain a homozygous plant from a heterozygous source.

A pollination control system and effective transfer of pollen from oneparent to the other offer improved plant breeding and an effectivemethod for producing hybrid canola seed and plants. For example, theogura cytoplasmic male sterility (cms) system, developed via protoplastfusion between radish (Raphanus sativus) and rapeseed (Brassica napus)is one of the most frequently used methods of hybrid production. Itprovides stable expression of the male sterility trait (Ogura, 1986),Pelletier, et al., (1983) and an effective nuclear restorer gene(Pellan-Dourme, et al., 1988).

In developing improved new Brassica hybrid varieties, breeders useself-incompatible (SI), cytoplasmic male sterile (CMS) and nuclear malesterile (NMS) Brassica plants as the female parent. In using theseplants, breeders are attempting to improve the efficiency of seedproduction and the quality of the F1 hybrids and to reduce the breedingcosts. When hybridization is conducted without using SI, CMS or NMSplants, it is more difficult to obtain and isolate the desired traits inthe progeny (F1 generation) because the parents are capable ofundergoing both cross-pollination and self-pollination. If one of theparents is a SI, CMS or NMS plant that is incapable of producing pollen,only cross pollination will occur. By eliminating the pollen of oneparental variety in a cross, a plant breeder is assured of obtaininghybrid seed of uniform quality, provided that the parents are of uniformquality and the breeder conducts a single cross.

In one instance, production of F1 hybrids includes crossing a CMSBrassica female parent with a pollen-producing male Brassica parent. Toreproduce effectively, however, the male parent of the F1 hybrid musthave a fertility restorer gene (Rf gene). The presence of a Rf genemeans that the F1 generation will not be completely or partiallysterile, so that either self-pollination or cross pollination may occur.Self pollination of the F1 generation to produce several subsequentgenerations is important to ensure that a desired trait is heritable andstable and that a new variety has been isolated.

An example of a Brassica plant which is cytoplasmic male sterile andused for breeding is ogura (OGU) cytoplasmic male sterile(Pellan-Delourme, et al., 1987). A fertility restorer for oguracytoplasmic male sterile plants has been transferred from Raphanussativus (radish) to Brassica by Instit. National de Recherche Agricole(INRA) in Rennes, France (Pelletier, et al., 1987). The restorer gene,Rf1 originating from radish, is described in WO 92/05251 and inDelourme, et al., (1991). Improved versions of this restorer have beendeveloped. For example, see, WO98/27806 oilseed Brassica containing animproved fertility restorer gene for ogura cytoplasmic male sterility,which is hereby incorporated by reference.

Other sources and refinements of CMS sterility in canola include thePolima cytoplasmic male sterile plant, as well as those of U.S. Pat. No.5,789,566, DNA sequence imparting cytoplasmic male sterility,mitochondrial genome, nuclear genome, mitochondria and plant containingsaid sequence and process for the preparation of hybrids; U.S. Pat. No.5,973,233 Cytoplasmic male sterility system production canola hybrids;and WO97/02737 Cytoplasmic male sterility system producing canolahybrids; EP Patent Application Number 0 599042A Methods for introducinga fertility restorer gene and for producing F1 hybrids of Brassicaplants thereby; U.S. Pat. No. 6,229,072 Cytoplasmic male sterilitysystem production canola hybrids; U.S. Pat. No. 4,658,085 Hybridizationusing cytoplasmic male sterility, cytoplasmic herbicide tolerance, andherbicide tolerance from nuclear genes; all of which are incorporatedherein.

Promising advanced breeding lines commonly are tested and compared toappropriate standards in environments representative of the commercialtarget area(s). The best lines are candidates for new commercial lines;and those still deficient in a few traits may be used as parents toproduce new populations for further selection.

For most traits the true genotypic value may be masked by otherconfounding plant traits or environmental factors. One method foridentifying a superior plant is to observe its performance relative toother experimental plants and to one or more widely grown standardlines. If a single observation is inconclusive, replicated observationsprovide a better estimate of the genetic worth.

A breeder uses various methods to help determine which plants should beselected from the segregating populations and ultimately which lineswill be used for commercialization. In addition to the knowledge of thegermplasm and other skills the breeder uses, a part of the selectionprocess is dependent on experimental design coupled with the use ofstatistical analysis. Experimental design and statistical analysis areused to help determine which plants, which family of plants, and finallywhich lines are significantly better or different for one or more traitsof interest. Experimental design methods are used to control error sothat differences between two lines can be more accurately determined.Statistical analysis includes the calculation of mean values,determination of the statistical significance of the sources ofvariation, and the calculation of the appropriate variance components.Five and one percent significance levels are customarily used todetermine whether a difference that occurs for a given trait is real oris due to the environment or experimental error.

Proper testing should detect any major faults and establish the level ofsuperiority or improvement over current lines. In addition to showingsuperior performance, there must be a demand for a new line that iscompatible with industry standards or which creates a new market. Theintroduction of a new line commonly will incur additional costs to theseed producer, the grower, the processor and the consumer, for specialadvertising and marketing, altered seed and commercial productionpractices, and new product utilization. The testing preceding release ofa new line should take into consideration research and development costsas well as technical superiority of the final line. For seed-propagatedlines, it must be feasible to produce seed easily and economically.Preferably residual heterozygosity should not exceed 5%.

These processes, which lead to the final step of marketing anddistribution, usually take approximately six to twelve years from thetime the first cross is made. Therefore, the development of new linessuch as that of the present invention is a time-consuming process thatrequires precise forward planning, efficient use of resources, and aminimum of changes in direction. Accordingly, significant technicalhuman intervention is required.

Further, as a result of the advances in sterility systems, lines aredeveloped that can be used as an open pollinated variety (i.e., apureline cultivar sold to the grower for planting) and/or as a sterileinbred (female) used in the production of F1 hybrid seed. In the lattercase, favorable combining ability with a restorer (male) would bedesirable. The resulting hybrid seed would then be sold to the growerfor planting.

Hybrid seed production of canola can be achieved using cytoplasmic malesterility. This type of hybrid production uses 3 inbred lines: arestorer line, an A line, and a B line. The restorer line, also calledthe R line, is used as the male in hybrid seed production. The restorerline has dominant nuclear genes, known as restorer genes, that areresponsible for hybrid fertility. The R line is crossed to the A line toproduce the F1 hybrid seed. The A line is male-sterile due to thecytoplasm and due to nonrestorer alleles in the nuclear genome. Becausethe A line is male sterile it cannot reproduce by itself. To reproducethe A line, a B line is developed. The B line, also called themaintainer line, is the genetic equivalent to the A line except that theB line has a normal cytoplasm and is therefore male fertile. The A lineis pollinated by the B line. The seed developed on the A line plants isharvested and its progeny are crossed with the R line to produce the F1hybrid seed.

The development of a canola hybrid in a canola plant breeding programinvolves three steps: (1) the selection of plants from various germplasmpools for initial breeding crosses; (2) the selfing of the selectedplants from the breeding crosses for several generations to produce aseries of inbred lines, which, although different from each other, breedtrue and are highly uniform; and (3) crossing the selected inbred lineswith different inbred lines to produce the hybrids. During theinbreeding process in canola, the vigor of the lines decreases. Vigor isrestored when two different inbred lines are crossed to produce thehybrid. An important consequence of the homozygosity and homogeneity ofthe inbred lines is that the hybrid between a defined pair of inbredswill always be the same. Once the inbreds that give a superior hybridhave been identified, the hybrid seed can be reproduced indefinitely aslong as the homogeneity of the inbred parents is maintained.

Combining ability of a line, as well as the performance of the line perse, is a factor in the selection of improved canola lines that may beused as inbreds. Combining ability refers to a line's contribution as aparent when crossed with other lines to form hybrids. The hybrids formedfor the purpose of selecting superior lines are designated test crosses.One way of measuring combining ability is by using breeding values.Breeding values are based on the overall mean of a number of testcrosses. This mean is then adjusted to remove environmental effects andit is adjusted for known genetic relationships among the lines.

Hybrid seed production requires inactivation of pollen produced by thefemale parent. Incomplete inactivation of the pollen provides thepotential for self-pollination. This inadvertently self-pollinated seedmay be unintentionally harvested and packaged with hybrid seed.Similarly, because the male parent is grown next to the female parent inthe field there is also the potential that the male selfed seed could beunintentionally harvested and packaged with the hybrid seed. Once theseed from the hybrid bag is planted, it is possible to identify andselect these self-pollinated plants. These self-pollinated plants willbe genetically equivalent to one of the inbred lines used to produce thehybrid. Though the possibility of inbreds being included in hybrid seedbags exists, the occurrence is rare because much care is taken to avoidsuch inclusions. These self-pollinated plants can be identified andselected by one skilled in the art, either through visual or molecularmethods.

Brassica napus canola plants, without any male sterility or selfincompatibility system, are recognized to commonly be self-fertile withapproximately 70 to 90 percent of the seed normally forming as theresult of self-pollination. The percentage of cross pollination may befurther enhanced when populations of recognized insect pollinators at agiven growing site are greater. Thus open pollination is often used incommercial canola production.

III. Definitions

Units, prefixes, and symbols may be denoted in their SI accepted form.

“Appropriate check”, as used herein, means a Brassica genotype whichprovides a basis for evaluation of the Sclerotinia resistance of anexperimental line. An appropriate check is grown under the sameenvironmental conditions, including disease pressure, as is theexperimental line, and is of approximately the same maturity as theexperimental line. For example, for spring canola, an appropriate checkis expected to mature within +/−10 days, usually +/−5 days, of theexperimental line. Maturity standards are well known to one of skill inthe art. An appropriate check is usually a widely-available orwidely-grown variety. The term “appropriate check” may actually reflectmultiple appropriate varieties. For example, for spring canolagenotypes, each of Pioneer Hi-Bred varieties 46A76 and 46A65 is anappropriate check; the mean performance of the two varieties is also anappropriate check. For winter canola genotypes, each of public linesColumbus and Express is an appropriate check, as is the mean performanceof the two varieties.

The term “canola” means a Brassica plant wherein the oil must containless than 2% erucic acid and the solid component of the seed mustcontain less than 30 micromoles of any one or any mixture of 3-butenylglucosinolate, 4-pentenyl glucosinolate, 2-hydroxy-3 butenylglucosinolate, and 2-hydroxy-4-pentenyl glucosinolate per gram ofair-dry, oil free solid.

The term “crossed” or “cross” in the context of this invention means thefusion of gametes via pollination to produce progeny (i.e., cells, seedsor plants). The term encompasses both sexual crosses (the pollination ofone plant by another) and selfing (self-pollination, i.e., when thepollen and ovule are from the same plant).

The term “field capacity” means that the top 4 inches of soil, orapproximately the top 4 inches of soil, are fully saturated withmoisture, but with no or little standing water.

The term “field resistance” means a resistance measured under fieldconditions. It reflects the resistance of the entire plant or populationof plants when exposed to the pest or pathogen in natural fieldconditions. Field resistance may be measured throughout thedevelopmental stages of a plant, and may be expressed in terms of effecton harvestable yield, or may reflect a targeted evaluation during thegrowth stage when the plant is most susceptible to disease development.

The term “genetically linked” refers to genetic loci that are in linkagedisequilibrium and statistically determined not to assort independently.Genetically linked loci assort dependently from 51% to 99% of the timeor any whole number value there between, preferably at least 60%, 70%,80%, 90%, 95% or 99%.

The term “inbred” as used herein refers to a homozygous plant or acollection of homozygous plants. Those of ordinary skill will understandthat some residual heterozygosity may exist in inbreds.

The term “introgression” refers to the introduction of a desired geneticlocus into at least one progeny plant via a sexual cross between parentplants and wherein at least one of the parent plants has the desiredgenetic locus within its genome.

The term “partial leaf resistance” means the extent of resistance toSclerotinia on the leaf when compared to the leaf reaction on asusceptible plant. With partial leaf resistance, the disease developsmore slowly on the plant, or to a lesser extent, than in a plant that issusceptible.

The term “marker” or “molecular marker” refers to a genetic locus usedas a point of reference when identifying genetically linked loci such asa QTL (quantitative trait loci). The term also refers to nucleic acidsequences complementary to the genomic sequences, such as nucleic acidsused as probes.

The term “partial stem resistance” or “stem resistance” means anincomplete resistance in the stem. It is the extent of resistance toSclerotinia on the stem when compared to the stem reaction on asusceptible plant. In partial stem resistance the disease develops moreslowly on the plant or to a lesser extent than in a plant that issusceptible. However, in a plant that has “partial stem resistance”, theplant becomes diseased (compare with complete resistance).

The term “complete resistance” means a resistant reaction in which anaspect of disease development, usually symptom expression or pathogenreproduction, is completely stopped (compare with partial resistance).

The term “recurrent selection” means a breeding system with theobjective of increasing the frequency of favorable genes of aquantitatively inherited characteristic by repeated hybridization andcycles of selection.

The term “maturity” or “days to maturity” means the number of days fromseeding to harvest. Maturity will vary considerably between and withingenotypes, depending on location, growing season and date of seeding.

The term “population” as used herein means an interbreeding group ofplants or a group of individuals that share a common gene pool. Apopulation may be homogenous (genetically uniform), such as an F1population created by crossing homozygous parents; or geneticallydiverse, such as the segregating progeny of a population created byselfing a heterozygous plant or by crossing heterozygous parents.Further, a homogenous population may be bred to be homozygous at almostall gene loci and produce a uniform population of true pureline progeny.

The term “population breeding” refers to improvement in a populationcarried through breeding while using the individuals from the samepopulation as parents. Population breeding can mean performing recurrentselection within a population.

The term “spring Brassica” or “spring canola” means a Brassica plantthat does not have a vernalization requirement.

The term “Sclerotinia conducive morphology” or “Sclerotinia conducivephenotype” means a Brassica phenotype in which Sclerotinia infection canestablish and develop more easily compared to a Brassica phenotype thatis less conducive to Sclerotinia infection. For example, a Sclerotiniaconducive morphology may include at least one of the followingmorphological traits: tight branching, low branching, extended durationof flowering, high petal retention, high degree of leaf retention, and apropensity to lodge or lean. These morphological traits provide a goodsource of the initial inoculum from the petals and increase moisturesurrounding the plant. In contrast, plants exemplifying morphologicaltraits that are less conducive to Sclerotinia infection may include atleast one of the following traits: low petal retention, petal-lessphenotype, good standability, less compact branching, high branching,and early leaf abscission. These morphological traits decrease inoculumfrom the petals as well as the level of moisture surrounding the plant.

The term “disease incidence” means the number of plants affected by adisease within a sample. It is typically presented as the percentage ofplants affected by the disease with respect to the total number ofplants in the sample.

The term “SSDI %” means percentage Sclerotinia Sclerotiorum DiseaseIncidence and is measured as the percentage of plants in a populationinfected with Sclerotinia sclerotiorum.

The term “SSDI” means a rating from 1 to 9 and measures the SclerotiniaSclerotiorum Disease Incidence under controlled extreme disease pressurefield research conditions as described in Example 7 and Table 4. SSDImeasures the percentage of plants in a population that are infected withSclerotinia sclerotiorum as compared to an appropriate check variety.For spring canola an appropriate check is Pioneer Hi-Bred variety 46A76and/or Pioneer Hi-Bred variety 46A65. For winter canola an appropriatecheck is Columbus and/or Express. For example, for spring canola, 5 rowsof test lines are sown between one row of Pioneer Hi-Bred variety 46A76on one side and Pioneer Hi-Bred variety 46A65 on the other. Typically,under extreme disease conditions, Pioneer Hi-Bred variety 46A76 has adisease incidence of 60% (SSDI %) and 46A65 has a disease incidence of70% (SSDI %), for an average of 65% (SSDI %). If in any particular testplot, the SSDI % average of Pioneer Hi-Bred variety 46A65 and PioneerHi-Bred variety 46A76 was not 65%, the scores of Pioneer Hi-Bred variety46A65 and Pioneer Hi-Bred variety 46A76 would be multiplied by a factorto bring them to an average of 65%. The scores of the test lines wouldalso be multiplied by this factor. The lines would then be given therating that corresponds with the SSDI % as found on Table 4. Forexample, if the mean of checks is 70%, a factor 65/70 would be used todecrease the percentage disease incidence measured on the experimentallines growing between the checks because of higher-than-targeted diseasepressure. Conversely, if the mean is 60%, a factor 65/60 would be usedto accordingly increase (adjust) SSDI % on the experimental linesbecause of the lower-than-targeted disease pressure. Although targeteddisease incidence is 65%, variation around the target is expected due tothe large sample of plants, environmental variation and variation ininoculum, therefore adjustments via checks enables comparison of lineswithin the nursery and prevents misclassification of the field reaction.Extreme disease pressure field research conditions as described inExample 7 were used extensively to produce the Sclerotinia resistantlines of the invention because conditions favorable for Sclerotinia donot occur in a predictable fashion in nature. Therefore in an effort toexpedite selections and to provide reproducible conditions, extremedisease pressure field research conditions were used.

The SSDI rating for trials, after adjustment for incidence, is furtheradjusted by taking into account the severity of the disease. Therefore,after adjusting the SSDI % as previously explained, SSDS adjustments aremade as well. The mean SSDS on infected plants of checks (scores of 1-8)is compared with that of the experimental entry. If the mean SSDS scorewas better on the experimental line (for example a rating of 3 on thechecks versus 4 on the experimental line) the SSDI % was adjusted bymultiplying by ¾. For example, if the SSDI % of the experimental linewas 20%, ¾ multiplied by 20%=15%. This corresponds to a rating of 7.5 onthe SSDI scale. Conversely, if the SSDS on the experimental entry was 2(more affected) versus 3 on the checks, the SSDI % would be multipliedby 3/2. For example, if the SSDI % was 20%, multiplied by 3/2 wouldequal 30%. This corresponds to a rating of 6.0 on the SSDI scale.

The term ‘extreme disease pressure field research conditions’ meanscontrolled disease research conditions as described in Example 7. Forexample, extreme disease pressure is generated with the application ofNiger seed carrier mimicking Sclerotinia-colonized petals. Naturalinoculum may be present in the field as a backup inoculum. The percentdisease incidence of the test plants are adjusted to running checks asdescribed above, and given an SSDI score of 1 to 9. However, under theseextreme conditions plants are more susceptible to Sclerotinia for atleast the following reasons: (1) under extreme disease pressure fieldresearch conditions, the plants are subjected to wetness provided bymisting irrigation which is favorable for Sclerotinia development, (2)under extreme disease pressure field research conditions the plants arein a semi-enclosed environment due to the artificial canopy whichensures continuous moist conditions favorable for Sclerotiniadevelopment, and (3) under extreme disease pressure field researchconditions there are six rows of different test plants in each plot,therefore any one row of test plants having a particular morphologicalphenotype may be surrounded by two different rows of plants withdifferent morphological phenotypes. Accordingly, any benefits from amorphological phenotype that is less conducive to Sclerotinia infection(for example high branching) are decreased because any one row may besurrounded by plants having a different morphological phenotype (forexample low branching). In contrast, a plant growing under natural fieldconditions is (1) not enclosed in an artificial canopy which ensurescontinuous moisture and (2) is grown in plots surrounded by plants withthe same morphological phenotype which allows all benefits from themorphology to be expressed. Accordingly, selections having a morphologythat is less conducive to Sclerotinia infection, for example highbranching, perform significantly better under natural field conditionscompared to extreme disease pressure field research conditions.

The term ‘natural field disease conditions’ means conditions in yieldplots in irrigated or non-irrigated fields. Infection is attained viamycelium from colonized petals. Yield plots provide a sample of theplant population that reflects natural conditions similar to farmers'fields. SSDI % is used to express the percentage of infected plants inreplicated trials. In addition to SSDI %, data on individual plants canbe collected to reflect severity (SSDS) on different scales (1-9 Pioneerscale and 0-5 Public scale). Other parameters that further quantify theimpact of disease, for example Sclerotinia sclerotiorum Field Severity(SSFS), as described below and shown in Table 2, can also be evaluated.SSFS can be informative especially under high natural field diseasepressure.

The term “disease severity” means the extent of damage to a plantresulting from infection by a pathogen. There are two scales used inthis invention to measure disease severity. The first is the PioneerHi-Bred scale from 1 to 9. The second is the scale used by researchersin public institutions and is referred to as the Public scale from 0-5.Both are described in Table 15. Some examples of their use are presentedin Table 2.

The term “SSDS” means Sclerotinia Sclerotiorum Disease Severity and is ameasurement of the extent of disease development on an infected plant.For example, it distinguishes between plants with minor symptoms versusdead plants. For the purposes of this invention, two rating scales areused: (1) The Pioneer SSDS rating scale ranges from 1 to 9 and isdescribed in Table 15; and (2) The Public Scientists' scale ranges from0 to 5 and is described in the footnote in Table 2.

The term “SSFS” means Sclerotinia Sclerotiorum Field Severity and is ameasurement of the product of disease incidence (SSDI %) and the extentto which infected plants were diseased under natural field conditions(SSDS). It is a measure of the fungal impact in the field and can bemore informative under high disease pressure, i.e. when diseaseincidence becomes significant in the field. It is calculated bymultiplying the SSDI % by the disease severity and dividing by 5,wherein the disease severity is rated 0 to 5, with 0 being no infectionand 5 being a dead plant as described in Table 2.

The term “quantitative trait locus” or “QTL” refers to segregatinggenetic factors that affect the variability in expression of aphenotypic trait.

TABLE 2 Field-collected Sclerotinia parameters SSDI % and SSDS and theirrelationship to derived parameters SSDI (research data) and SSFS(natural data). SSDI SSFS Based on Field adjusted SSDI % severity underextreme based on both SSDS disease SSDI % and SSDI % Disease pressurefield SSDS Disease severity of research used in natural Trait Incidenceaffected plants conditions field conditions Scale 0-100% Pioneer SSDS1-9 0-100% scale Conversion of % field impact - 1 = dead SSDI % andquantifies 9 = no disease adjustment for damage in the field Publicscale checks irrespective of 0 = no disease disease 5 = dead plantpressure Usage General General Pioneer only General Adjustments N/A N/AAdjusted to Unadjusted checks Examples: Different combinations ofdisease incidence and Pioneer disease SSDS Public severity scale scaleExample 1 80 1 5.0 1.0 (80) 80 Example 2 80 5 2.0 2.6 (64) 32 Example 350 5 2.00 5.0 (40) 20 Example 4 30 7 1.0 7.3 (17) 6 Example 5 10 8 1.08.5 (5)  2

SSDI % is the percentage of plants in a population infected withSclerotinia.

SSDS is a rating of the extent of disease development on an affectedplant. Two scales are used in the invention. The Pioneer SSDS scaleranges from 1 (dead) to 9 (no disease) and the Public scale ranges from0 (no disease) to 5 (dead) plant. For details of the Pioneer SSDS scale,see Table 15. The Public scale is provided as follows: 0=no disease;1=superficial lesions or small branch affected; 2=large branch dead;3=main stem at least 50% girdled; 4=main stem girdled but plant producedgood seed; 5=main stem girdled, much reduced yield.

SSDI is a rating of 1 to 9 as described on Table 4, and adjusted to theSSDI % of the check varieties 46A65 and/or 46A76 for spring canola, andcheck varieties Express and/or Columbus for winter canola. This ratingis used only under controlled extreme disease pressure field researchconditions. It is calculated by multiplying the observed SSDI % byFactor X, where Factor X is the factor that brings the average SSDI % ofthe appropriate checks to 65%. Adjustment for severity is done afterincidence adjustment. The SSDI is then calculated according to the scaleon Table 4. For examples 1 to 5, assumptions are that mean SSDI % onchecks=65% and the mean SSDS on checks=4 to calculate SSDI values.

SSFS is a measure of both disease incidence and severity under naturaldisease pressure in the field. It is calculated as follows: SSFS=[SSDI%×SSDS (0-5 scale)]÷5

IV. Examples

Sclerotinia stem rot develops in canola via colonized petals in extendedmoist conditions at flowering. Dropped petals enable Sclerotinia toinfect leaves of canola leading towards the stems. The fungus causesSclerotinia stem rot. The plant dies prematurely which results in ayield loss of approximately 50%.

Canola is susceptible to Sclerotinia stem rot. In years with extendedwet periods, damage to canola can be very significant. To reduce orprevent that damage, farmers generally apply one or two fungicideapplications, depending on the duration of the wet period.

Example 1 Determining the Performance of Canola Checks Under Low,Moderate, High, Very High and Extreme Disease Field Research ConditionsMethods and Materials

In an effort to determine the level of Sclerotinia tolerance incurrently available spring canola cultivars under low, moderate, highand very high Sclerotinia conditions, data was collected from naturalfield conditions over many years, including public yield plots. The datais summarized in Table 3. Data for 44A89 and 46A65 came from a fivereplication-natural trial in Minnesota in 2001 (Jurke and Fernando,2003). The data for Pioneer Hi-Bred variety 46A76 is an estimate basedon the reaction of similar entries in the same Minnesota trial as wellas North Dakota data from 2003. The data for the performance of thecanola checks under extreme disease conditions was generated in thisstudy.

Winter canola lines Columbus and Express were included in extremedisease pressure research trials as running checks. As shown in Table 4and Table 10c, these are susceptible or moderately susceptible lines.

Extreme disease conditions are rare but may occur, and the goal was toscreen lines under such conditions. Extreme disease pressure occurs with20-30 days of continuous wetness and temperatures averaging 20 to 25° C.Plants infected with Sclerotinia under extreme conditions usuallyrequire two fungicide applications to withstand the disease. On average,fungicides provide 10-14 days of protection per application. Screeningselections under extreme conditions ensures the selections can withstandtypical disease pressure.

Because extreme disease conditions occur rarely in nature, artificialextreme conditions were generated in the field as described in Example7. This included artificial inoculum in the form ofSclerotinia-colonized Niger seed, the use of irrigation, and the use ofa netting to maintain a moist environment. Pioneer Hi-Bred varieties44A89, 46A65 and 46A76 were tested. Less susceptible 46A65 and 46A76were used as running checks to monitor disease levels and determineperformance (SSDI).

In an effort to determine the effect of fungicide spraying onSclerotinia-infected fields, data from yield plots under natural fieldconditions and inoculum, and sprayed with Lance™ fungicide during 30%flowering, was collected and summarized.

Results

Table 3 shows the performance of spring canola checks under variousconditions. Screening for disease incidence (i.e., the percentage ofplants infected with Sclerotinia) was the primary goal.

Previous field data indicated that typical moderate or high fielddisease pressure results in 20-50% disease incidence on 46A65 and 10-40%disease incidence on 46A76 (Table 3). However, under extreme conditionsas shown in Table 3, the percentage of disease incidence on PioneerHi-Bred variety 46A65 is approximately 70% and the percentage of diseaseincidence on Pioneer Hi-Bred variety 46A76 is 60%. If the weatherbecomes unfavorable for Sclerotinia infection, the canola checks areless affected and the disease incidence on partially resistant materialsis proportionally minimized or eliminated. Since most field situationsare based on less than extreme disease pressure, the checks and thedeveloped lines will normally be less affected than shown in Table 4.

Screening for disease incidence under extreme disease pressure andagainst running checks every six rows under misting irrigation, was alsodone as described in Example 7. This extreme disease pressure researchfield data is presented as an SSDI rating of 1 to 9 on Table 4.Typically, 5 rows of test lines were sown between one row of PioneerHi-Bred variety 46A76 on one side and one row of Pioneer Hi-Bred variety46A65 on the other. Under extreme disease conditions, Pioneer Hi-Bredvariety 46A76 has a disease incidence of 60% (SSDI %) and PioneerHi-Bred variety 46A65 has a disease incidence of 70% (SSDI %), for anaverage of 65% (SSDI %). If in any particular test plot, the SSDI %average of Pioneer Hi-Bred varieties 46A65 and 46A76 was not 65%, thescores of Pioneer Hi-Bred variety 46A65 and Pioneer Hi-Bred variety46A76 would be multiplied by a factor to bring them to an average of65%. The scores of the test lines would also be multiplied by thisfactor. After making the severity adjustment as described in thedefinition of SSDI, the lines would then be given the rating thatcorresponds with the SSDI as found on Table 4.

TABLE 3 Variation in natural Sclerotinia field reaction of currentlyavailable spring canola (SSDI %) Disease Pressure Category Variety LowModerate High Very High Extreme* Highly susceptible 44A89 10-30  30-6050-80 70-90  80-100 Susceptible 46A65 0-10 20-30 30-50 40-60 70Moderately susceptible 46A76 0-10 10-20 20-40 30-50 60 % of occurrence -field** 40 30 20 8-9 1-2 *Extreme disease pressure is used for coreresearch and development as described in Example 7. **Estimate offrequency of each infection level in farmers' fields in Western Canada,North Dakota and Minnesota.

TABLE 4 Measuring field performance under extreme disease pressure(research trials) Disease Rating incidence Spring Winter SSDI** CategorySSDI % Checks Checks 1.0 Highly susceptible ≧80 44A89, Panther Westar1.1-2.0 Susceptible 79-70 46A65 = 2 Columbus = 2 2.1-3.0 Moderatelysusceptible 69-60 46A76 = 3 Express = 3 3.1-4.0 59-50 4.1-5.0 Moderatelyresistant 49-40 5.1-6.0 39-30 6.1-7.0 Resistant 29-20 7.1-8.0 19-108.1-9.0 Highly resistant 9-0 *SSDI % Sclerotinia Sclerotiorum DiseaseIncidence % **SSDI Sclerotinia Sclerotiorum Disease Incidence rating asadjusted for incidence and severity on checks 46A65/46A76 for springcanola and Express/Columbus for winter canola under extreme diseasepressure (research trials).

Table 5 shows that fungicide applications reduce the effect ofSclerotinia on Brassica and can be used as an indirect measure ofimprovements in performance against Sclerotinia. Table 5 shows theeffect of one fungicide application in replicated yield plots undernatural infection at Morden and Carman, Manitoba in 2004. As seen inTable 5, under conditions of lower disease pressure, near-completecontrol of Sclerotinia is achieved with a single fungicide application,except for highly susceptible materials. Fungicide efficacy on materialwith a rating of 1 (HS) is lower than the fungicide efficacy on materialrated 2 or 3 (S or MS).

TABLE 5 Sclerotinia infection (SSDI %) on sprayed* and unsprayed checksin yield plots under natural conditions-in Morden, Manitoba and Carman,Manitoba locations in 2004 Variety Category Morden Carman Mean Fungicideapplication 44A89 HS 30.7 22.5 27 Unsprayed 44A89 HS 8.0 19.5 14 Sprayed46A65 S 11.3 11.5 11 Unsprayed 46A65 S 0.0 2.0 1 Sprayed 46A76 MS 11.39.5 10 Unsprayed 46A76 MS 2.7 1.0 2 Sprayed *Lance ™ (boscalid) - BASFregistered fungicide for control of Sclerotinia HS = highly susceptible;S = susceptible; MS = moderately susceptible

Table 6 shows the results from the field trials in NorthDakota/Minnesota conducted in 2003. Most currently commerciallyavailable canola varieties are rated 1 or 2 based on Pioneer's SSDIrating of 1 to 9 as described in Table 2. Some rare varieties are rated3 and are more effectively protected by fungicides. For example, Table 6shows that Hyola 357, having a Sclerotinia rating of approximately 2,had 69% incidence in the North Dakota fungicide trials. Afterapplication of the best fungicide, the incidence was reduced to 44%.Table 6 also shows that Invigor2663, having a Sclerotinia rating of 1 to2, had 22.3% incidence in the Minnesota trial, where disease pressurewas low. After application of the fungicide, the incidence was reducedto 5%.

TABLE 6 Canola variety performance in the Sclerotinia screening andfungicide trials at same locations, North Dakota State UniversityCarrington Research/Extension Center and University of Minnesota RedLake Falls, 2003.* North Dakota Minnesota Disease Disease Field DiseaseDisease Field Variety Incidence Severity Severity Incidence SeveritySeverity Hyola 357-untreated 69 2.3 32.1 fungicide trial Hyola357-treated 44.0 1.9 16.6 fungicide trial Endura (Boscalid) Hyola357-variety trial 60.5 2.6 30.9 18 4.8 17.3 InVigor2663-variety trial61.5 2.8 34.4 30.0 4.5 27.0 InVigor2663-untreated 22.3 4.0 17.9fungicide trial InVigor2663-treated 5.0 3.3 3.1 fungicide trial Endura(Boscalid) Based on disease incidence and Table 3, the North Dakotatrial can be classified as very high to extreme disease pressure.Outcome of the fungicide trial at North Dakota is comparable withextreme disease pressure on the Pioneer SSDI scale of 1-9 and indicatesperformance of best fungicide in protection of a susceptible variety.Endura ™ (Boscalid ™) provided the highest level of the protection inthe trial against a number of other fungicides under this pressure.*From 2003 Evaluations for fungicides for Control of Sclerotinia StemRot of Canola in North Dakota and Minnesota, NDSU Extension ServiceApril 2004.

Example 2 Developing Resistance to Sclerotinia-Population T Development

The target of the research effort was to replace fungicide treatment ofcanola with Sclerotinia-resistant varieties. The strategy was to usenaturally available sources with partial resistance and pyramid thesewith disease-avoiding morphological traits through recurrent selectionwithin a population, in order to attain a very high level of partialresistance. Once pyramided in a disease-avoiding background, resistancewould be complete if it were built to sustain the maximum length ofexposure to the disease, starting from petal drop to the end offlowering, and therefore would withstand the pathogen withoutsignificant damage to the plant.

Disease-avoiding morphological traits include, for example, goodstandability and stiff stalk (stem), later maturity, high branching,lower petal retention and rapid leaf abscission. Physiological traitsare primarily strong partial stem resistance which may be associatedwith some leaf resistance. Thus, disease development which is reduced bymorphological traits is further reduced where stem and/or leafresistance is present. The overall impact of the fungus on the stem isdecreased or minimized when a strong partial resistance in stems iscombined with morphological traits that reduce the impact of thedisease. In the absence of favorable morphological traits, overallperformance is reduced but still significantly better than canolachecks.

Extreme disease pressure occurs with prolonged periods of favorableweather conditions for Sclerotinia infection. Typically, this occurswith temperatures between 20 and 25° C. and relative humidity of greaterthan 80%. Under these conditions, optimal plant infection occurs (Heran,et al., 1999). In addition to humidity, another indicator of wet plantcanopy is free moisture or absolute moisture on plants.

Methods and Materials

Tables 7 and 8 describe the components used in developing fieldresistance in Population T and the methods used to develop and screenfor field resistance.

Starting in 1986, Brassica napus rapeseed germplasm from governmentagencies in the United States (United States Department of Agriculture),Japan (Ministry of Fishery and Natural Resources) and Canada (PlantGenetic Resources) was acquired. Rapeseed is high in glucosinolates anderucic acid and therefore is not canola quality. It is referred to asdouble high. In contrast, Brassica napus canola quality is low in erucicacid and low in glucosinolates and is also referred to as double low. Itis defined as a Brassica plant wherein the oil must contain less than 2%erucic acid and the solid component of the seed must contain less than30 micromoles of any one or any mixture of 3-butenyl glucosinolate,4-pentenyl glucosinolate, 2-hydroxy-3 butenyl glucosinolate, and2-hydroxy-4-pentenyl glucosinolate per gram of air-dry, oil free solid.

The acquired rapeseed germplasm was characterized for stem reaction toSclerotinia (Table 7). A number of partially stem resistant rapeseedselections were crossed with canola quality lines, and progenies withpartial stem resistance and canola quality traits (low erucic and/or lowglucosinolates) were extracted. Canola quality lines that exhibitedpartial stem resistance were used for further research and populationdevelopment. The sources of Sclerotinia resistance used in developmentof improved spring canola lines are listed in Table 7. Introductionsfrom the USDA (US) and MAFF (Japan) were also used in development ofimproved winter canola lines.

In 1991, Pioneer started a population breeding program to recombinecanola materials with physiological partial stem resistance and fieldmorphological avoidance. Population T was assembled. Details of Cycles 0to 10 are shown in Table 8. Each cycle is described in a row in thetable. Each row describes the material used, intercrossing to generateS₀, seed increases and greenhouse selection on S₀ to generate S₁,followed by S₁ testing in the field and selection including agronomy andquality analysis. The first four cycles employed a closed population asdescribed in Table 8. Further variability for physiological partial stemresistance was injected into Cycles 5, 6, 7 and 8 (Table 8). The lineswere tested following the greenhouse and field methods of selection asdescribed in Examples 6 and 7. Progenies exhibiting high levels ofresistance were extracted and continuous improvement was observed in thefield (FIG. 1). Following the method of Example 6, only greenhousestem-selected progenies were advanced to the field.

TABLE 7 Population T development - Rapeseed components that wereconverted to canola and used in the development of Population T (IndoorStem Lesion length and SSDS data on original lines or their springselections (USDA) vs. susceptible checks) Lesion SSDS Lesion Length 1-9Rapeseed source Receiving Introgressing Length SSDS SusceptibleSusceptible Institution variety name year year Millimeter 1-9 CheckCheck The JAP 3-1-1 1988 1991 33 4.4  72* 2.0 Ministry of Ro (USSR) 61988 1991 28 4.2 48 1.0 Agriculture, Genkai 1988 1999 60 1.4 72 2.0Forestry Minami kyuushuu 17 1988 2000 10 5.4 48 1.0 and Fisheries ofJapan MAFF USDA PI470079 1988 2001 32 4.7  109** 1.1 North PI469955 19882001 27 4.5 120  1.0 Central PI469830 1988 2002 35 5.0 104  1.0 RegionalPlant Introduction Station Iowa State University Plant PGR 8487 19862002 27 5.2 85 1.2 Genetic PGR 8488 1986 2002 32 4.8 85 1.2 ResourcesPGR 8490 1986 2002 33 4.1 85 1.2 Canada PGR 8492 1986 2002 34 4.6 85 1.2PGR 8493 1986 2002 23 6.3 85 1.2 *Susceptible check for Japanesematerial spring canola Westar **Susceptible check for USDA and PGRmaterials was Pioneer's spring line NS1602

TABLE 8 Population T development through S1 recurrent selection methodwith modifications. Comments S₀ to S₁ S₁ Additional MATERIAL CrossingGrow GH Characterization Selection at S1 Partially selections Test FieldTest S₁ Agronomy and POPULATION Resistant Intercross for Select Fromselected S₀ Type of the test Quality POP T SOURCE S₀ S₀ to S₁Sclerotinia Year Disease pressure Field Checks Analysis CYCLE 0 JAP3-1-1 12 lines 500 GH 1993-1995 Stem test ESTABLISHING Ro (USSR) 6 TwiceStem inoculation population Half diallel 500 CYCLE 1 Closed 33 500 1501996 Natural NS1602 S₁ Population S₁ lines Moderate to high NS1604 NIRselection Pressure Not running Low glucosinolates CYCLE 2 Closed 30 500 90 1997 Moderate 46A65 S₁ Population S₁ Pressure NS1604 NIR selectionLines Pairs of checks Low glucosinolates CYCLE 3 Closed 12 500 150 1998Moderate 46A65 S₁ Population S₁ Pressure NS1604 NIR selection LinesPairs of checks Low glucosinolates CYCLE 4 Closed 15 3,000 1,100 1999Extreme 46A65 S₁ Population S₁ unreplicated NS1604 NIR selection LinesLow glucosinolates CYCLE 5 Open population 62 S₁s 3,000 176 2000 Extreme46A65 S₁ BC1 +15 Genkai 2 reps NS1604 Agronomic GENKAI lines NIRselection Low glucosinolates CYCLE 6 Open 41 C5 +9 F1 3,000 150 2001Extreme 46A65 S₁ Population introgressions 2 reps 46A76 Agronomic BC1NIR selection MINAMI Low KYUUSHUU 17 glucosinolates Blackleg selectionCycle 7 Open 24 S₁s cross 3,000 600 2002 Extreme 46A65 S₁ Populationwith F1 2 reps 46A76 Agronomic BC1 introgression NIR selection PI469955Low PI470079 glucosinolates Cycle 8 Open 30 S₁ 4,000 600 2003 Extreme46A65 S₁ Population 2 reps 46A76 Agronomic BC0 NIR selection PGR8487 LowPGR8488 glucosinolates PGR8490 PGR8492 PGR8493 PI469830 Cycle 9 Closed24 S₁s 4,000 600 2004 Extreme 46A65 S₁ Population 2 reps 46A76 AgronomicNIR selection Low glucosinolates Cycle 10 Closed Population 62 S1s 4,000390 2005 Extreme 46A65 S1 2 reps 46A76 Agronomic NIR selection Table 8Notes: Replicated testing occurred since Cycle 6. “Material” includesgenetic backgrounds used in development of source materials of canolaquality Closed Population = Population development based only onprogenies from previous cycle, no new sources introduced Open population= Population development based on progenies from the previous cycle aswell as new sources previously not present in the population.Introgression = Introduction of a new source into the population;associated with open population GH = greenhouse The letter F is usuallyused in breeding (in pedigree breeding) and represents filial/progenygeneration, F1 being first-generation seed or plant from the cross. Srefers to selfing, F1 is S0 or no selfing. In this way one candifferentiate progenies from population (S) vs pedigree breeding (F). S0is F1 and is often used in population breeding to indicate a number ofselfing generations S1 in population breeding is equivalent to F2 inpedigree breeding Examples of different approaches as follows: Year 0lntercrossing source material to produce S0, S0 Selfing/selecting, S1field selection in closed population Year 1 lntercrossing of S1s toproduce S0, S0 Selfing/selecting, S1 field selection; example of closedCycle (Cycles 1-4 and Cycle 9, 10) Year 2 lntercrossing of S1 from Year1 and new sources - BC0 approach (Cycle 8) or example of open cycle(Cycle 8) lntercrossing of S1 from Year 1 and new sources alreadycrossed with Pop T - BC1 approach (Cycles 5, 6, 7); example of opencycle (Cycles 5, 6, 7)

Results

FIG. 1 is a histogram of lines generated in Cycles 5 to 10 of PopulationT, showing the progress toward Sclerotinia resistance made under extremedisease pressure field research conditions, as measured against checksin Cycles 5 to 10. With each year of Population T improvement, thepercentage of disease incidence dropped and the population mean improvedon the SSDI scale of 1-9. The Y-axis represents the frequency ofprogenies and the X-axis shows the Sclerotinia resistance rating on SSDIscale of 1-9 as described in Table 4. FIG. 1 shows that the mean and themode of the population for each cycle have continually improved. Forexample, the mode of the population improved from 2.5 in Cycle 5, to 5.5in Cycle 8, to 7.5 in Cycle 10. In addition, individual selections inCycles 7, 8, and 9 had rating of 7.5, 8.0, and 8.5. Progenies exhibitinghigh levels of resistance were extracted and continuous improvement wasobserved in the field.

The extent of disease incidence in tests represented by FIG. 1 wasmeasured under extreme disease pressure field research conditions. Thisis the highest possible disease pressure in natural field environments.This level of disease seldom occurs in farmers' fields (Table 3).Accordingly, it is expected that materials selected in this test willperform much better under lower and more typical disease pressure. Forexample, under typical moderate field disease pressure, the diseaseincidence for Pioneer Hi-Bred varieties 46A65 and 46A76 is generally20-30% and 10-20% respectively (Table 3). However, under extreme diseasepressure the level of disease incidence is 70% and 60% respectively(Table 3). A plant with partial field resistance exhibits (i) reduceddisease development on the plant, (ii) significantly delayed onset ofdisease and (iii) resistance of disease development for a longer timewhen inoculum is in direct contact with the stem. If disease-favorableconditions persist, a significant reduction in the effect of disease isobserved in partially resistant materials (Table 3 and FIG. 1).

Tables 9a, 9b and 9c describe some of the lines with improved fieldresistance and show their performance in the field under naturalconditions in tests conducted by independent third parties. The 2004data on SSDI % were generated in Manitoba in open-field trials undermoderate disease pressure (as determined by the performance of 2 out of3 checks). Some of the same lines were tested at the University ofMinnesota (MN) under high pressure in 2003 (2 out of 2 checks) and inNorth Dakota (ND) under very high to extreme pressure in 2003 as perTable 3. All field resistant lines possess levels of partial stemresistance significantly higher than checks. Note that across the fourlocations, the performance of each of the five listed lines was wellwithin the present claim requirements, i.e., having an averageSclerotinia Sclerotiorum Disease Incidence (SSDI %) score which is lessthan about 60% of the SSDI % score of Pioneer Hi-Bred variety 46A76under the same environmental and disease conditions in the field.

Selections of Population T were also tested under extreme diseasepressure field research conditions (Table 10a and Table 10b). Selection02SN41269 was introduced into Cycle 8. Cycle 8 selection 03SN40441 wasused to develop Cycle 9 in combination with 03SN40341 and 22 S1 linesfrom previous Cycle 8 as outlined in Table 8. These three lines (i.e.02SN41269, 03SN40341 and 03SN40441) were used broadly in crossing, andPopulation T progenies trace back part of their genetic resistance toone or more of them.

Table 10a shows the results of three tests (two in 2004 and one in 2003)under extreme disease pressure field research conditions for 03SN40341,03SN40441 and 02SN41269. On average, 03SN40341, 03SN40441 and 02SN41269had a disease incidence (SSDI %) of 39%, 39% and 44% respectively,compared to 46A76. Under these extreme conditions plants are moresusceptible to Sclerotinia for at least the reasons discussed above inthe definition of “extreme disease pressure field research conditions.”In contrast, a plant growing under natural field conditions (1) is notenclosed in an artificial canopy which ensures continuous moisture and(2) is grown in plots surrounded by plants with the same morphologicalphenotype which allows all benefits from the morphology to be expressed.Accordingly, selections having a morphology that is less conducive toSclerotinia infection, for example high branching, perform significantlybetter under natural field conditions compared to extreme diseasepressure field research conditions. For example, on average, undernatural field conditions, 03SN40341, 03SN40441 and 02SN41269 had adisease incidence of 23.2%, 13.7% and 49.1% respectively, compared to46A76, as shown in Table 9a. Accordingly, 02SN41269 has a morphologythat is more conducive to Sclerotinia infection than 03SN40341 and03SN40441. 02SN41269 is more prone to lodging compared to 03SN40341 and03SN40441 (FIG. 2A). FIG. 2A reveals that 02SN41269 double haploid line04DHS12921 as well as doubled haploid 04DHS11319 are also prone tolodging more than check 46A65 and other tested material. Low resistanceto lodging can compromise Sclerotinia performance in natural data setsespecially with a lot of moisture or excessive irrigation such as atNDSU-Carrington 2005. Research data sets bypass lodging resistance andprovide scores that reflect a potential of genetic resistance incombination with morphology.

The effect of morphology under extreme disease pressure field researchconditions in comparison to natural field conditions can be demonstratedusing the check varieties 46A76, 46A65 and 44A89 as examples. 46A76 isone of the least susceptible varieties in the Sclerotinia Variety Trialsin North Dakota and Minnesota University Extension trials (Tables 9a and9b, 2003 data). 46A76 has a morphology that is less conducive toSclerotinia infection compared to 46A65 or 44A89. The morphologyincludes features such as high branching and very good standability.Considering SSDI % scores under extreme disease pressure field researchconditions (Table 10a), 44A89 is 115% of 46A76, and 46A65 is 98% of46A76. However, in natural trials in 2004 (Table 9a), 44A89 is 232% of46A76 and 46A65 is 199% of 46A76. This clearly shows that 46A76 is ahigh standard for measurement of field resistance, especially undernatural field conditions, in comparison with other canola checks. Thisalso shows the difference between natural field results and extremedisease pressure field research results. Extreme disease pressure fieldresearch conditions challenge all genotypes and represent the worst-casescenario, i.e. that which occurs normally in only 1 to 2% of naturalfield environments. (See, Table 3) A line which performs well underthese extreme conditions is expected to perform at least as well in anatural field environment.

Inclusion of check varieties such as 44A89 and 46A76 aids in evaluationof the testing environment and of the reliability of the data generated.For example, Tables 9b and 9c compare results obtained on control linesat North Dakota and Minnesota testing sites in 2001, 2003 and 2005.These sites are extraction of higher-disease-pressure sites, excludingdata sets from years with trace levels of disease (Bradley, et al.,2006). The low level of disease incidence for 46A76 in 2005(NDSU-Carrington) indicates that environmental conditions may havecomplicated the disease scoring as suggested by researcher Bob Hanson(NDSU). In this instance, excessive irrigation led to both excessivelodging on earlier lines and delayed maturity on later lines, favoringlater-maturing lines such as 46A76. Tables 9b and 9c suggest that thedata collected at the NDSU-Carrington 2005 site are inconsistent withthat generated at the same site as well as Minnesota sites in two prioryears (see also, Bradley, et al., 2006).

FIG. 2 provides data on multiple trials of spring lines under bothextreme disease pressure and natural field conditions. Common extremedisease pressure field research data was collected at up to four tests.Natural field data was collected at up to three locations including theNorth Dakota site discussed above (2005 data). When the results from thesingle aberrant trial are removed (FIGS. 2D and 2E), all tested linesclearly performed well within the target range, i.e. with an SSDI %score less than 60% of the SSDI % score of Pioneer variety 46A76, or ofthe SSDI % score of Pioneer variety 46A65, or of the mean SSDI % scoreof the two varieties, under the same environmental and diseaseconditions in the field.

Doubled haploid lines representing 02SN41269 (2 lines), 03SN40441 and03SN40341 were extracted and characterized under extreme diseasepressure field research conditions (Table 10b). The doubled haploidlines were produced by methods known to those skilled in the art; forexample see Swanson, et al., (1987); Mollers, et al., (1994); Hansen, etal., (1996); U.S. Pat. No. 6,200,808; and Canadian Patent Number2,145,833. The four doubled haploid lines had a SSDI rating of 6.6 to7.3. This compares well against the check, Pioneer Hi-Bred variety46A76, with a rating of 3.8. The doubled haploid lines havemorphological phenotypes similar to their parental lines, and they arelikely to perform better under natural conditions as well.

Table 10c provides performance data for F3 winter canola lines, 2005.The data in this Table originate from a 2005 field trial performed atTavistock (Ontario, Canada) that was replicated three times. Extremedisease pressure was used to generate this research data set.

The field reaction of the submissions was superior to that of thechecks, Express and Columbus, even under weather conditions whichresulted in disease pressure higher than the target. Lodging scores onwinter canola selections collected at Soest (Germany) have enabledselection of resistant material with good standability.

Table 11a shows pedigrees of five heterozygous spring canola selections(02SN41269, 03SN40341, 03SN40441, 04SN41433, and 04SN41415) and theirhomozygous doubled haploid progenies. Since all selections originatefrom a single plant (deriving 100-500 seeds for the first year of fieldtesting) they needed to be further selfed and increased as bulks forfurther field testing and seed submissions.

In summary, as shown in the histogram of FIG. 1, the breeding andselection efforts over six years (2000-2005) stemming from 15 years ofresearch (1991 to 2005) have resulted in improved spring canola lines.Under natural field conditions these lines have an average SclerotiniaSclerotiorum Disease Incidence (SSDI %) score which is less than about60% of the SSDI % score of Pioneer Hi-Bred variety 46A76, or of the SSDI% score of Pioneer Hi-Bred variety 46A65, or of the mean SSDI % score ofthe two varieties, under the same environmental and disease conditionsin the field. Table 9a shows that Pioneer Hi-Bred variety 46A76 is oneof the least susceptible canola checks in the field (rated a 3 on theSSDI scale, while most current canola products are rated 1 or 2), andrepresents a very challenging target against which to compare newmaterials. In addition, Lance™ or Endura™ are considered the mostefficacious fungicides on the market. This combination of the leastsensitive canola check and most efficacious fungicide sets the bar veryhigh. As seen from the histogram of FIG. 1, Cycle 10 of Population T hadan SSDI mode of 7.5, with certain individuals having a rating of 8 or8.5. According to the rating scale in Table 4 and the results shown inFIG. 1, lines having SSDI ratings of 5, 6, 7 or 8 have been developed.They are categorized as moderately resistant (rating of 5 or 6) andresistant (rating of 7 or 8).

TABLE 9a Summary of Sclerotinia natural field data results on thedeveloped Sclerotinia resistant materials in 2003/2004 2004 2004 20032003 2003/004 2003/2004 2003/2004 2003 2003 VARIETY PEDIGREE SSDI % SSDI% SSDI % SSDI % SSDI % SSDI % SSFS* SSFS SSFS SSFS MB MB 4 % of % of2003/2004 (Morden) (Carman) ND** MN locations 46A76 ND MN Mean 46A7602SN40680 Cycle 7-BN3 0.6 1.4 18.5 4.0 6.1 23.4 8.4 0.6 4.5 20.602SN40209 Cycle 7-BN4 4.9 17.4 22.0 0.0 11.1 42.3 10.1 0 5.1 23.102SN41269 02SN40102-BN1 4.0 1.4 39.0 7.0 12.9 49.1 16.6 3.5 10.1 46.046A76 MS check 18.3 14.6 51.0 21.0 26.2 100.0 25.6 18.1 21.9 100.0 44A89HS check 42.2 35.4 90.0 76.0 60.9 232.4 64.8 76.0 70.4 322.2 2 % of 2004only locations 46A76 03SN40441 Cycle 8 1.7 2.8 2.2 13.7 03SN40341 Cycle8 3.2 4.4 3.8 23.2 46A65 S check 33.3 32.0 32.6 198.8 *Field severity atND and MN is calculated by multiplying disease incidence (SSDI %) withdisease severity on infected plants and dividing by 5 (SSFS = [SSDI % ×SSDS (0-5 scale)] ÷ 5). A lower severity ‘1’ versus dead plant ‘5’ willsignificantly decrease overall impact of the disease as seen bycomparing 44A89 vs. 02SN40680. ND = North Dakota MN = Minnesota MB =Manitoba

TABLE 9b NDSU and University of Minnesota natural data on canola varietyfield reaction to Sclerotinia (% disease incidence) 2001/2003/2005* 20012001 2003 2003 2005 2001-2003 ALL Cultivar Carrington Red Lake FallsCarrington¹ Red Lake Falls² Carrington Mean Mean Hylite201** 14.7 1565.0 4.0 41 25 28 Hyola401 20.7 33 55.0 11.0 54.5 30 35 46A76 22.7 34.751.0 21.0 16.0 32 29 Hyola357 34.0 41 60.5 18.0 55.5 38 42 LG3455 41.341 52.5 29.0 30 41 39 44A89 36.0 73 90.0 76.0 49.5 69 65 *Data generatedby Bob Hanson (NDSU) and Dave Legare (University of Minnesota)**Apetalous canola ¹Carrington (North Dakota) 2003 data corresponds toTable 9a 2003 ND data ²Red Lake Falls (Minnesota) 2003 data correspondsto Table 9a 2003 MN data

TABLE 9c NDSU (Carrington) and University of Minnesota (Red Lake Falls)natural data on canola variety field reaction to Sclerotinia (diseaseincidence) 2001/2003*, expressed as % of 46A76 2001 Carrington 2001Sclerotinia Red Lake 2003 2003 2005 2001-2003 ALL Cultivar incidence (%)Falls Carrington Red Lake Falls Carrington Mean Mean Hylite201** 65 42127 19 256 63 102 Hyola401 91 96 108 52 341 87 138 46A76 100 100 100 100100 100 100 Hyola357 150 118 119 86 347 118 164 LG3455 182 119 103 138188 135 146 44A89 159 211 176 362 309 227 243 *Data generated by BobHanson (NDSU) and Dave Legare (University of Minnesota) **Apetalouscanola

TABLE 10a Summary of extreme disease pressure research data 2003-2004sources (SSDI) 3 tests 2004 2004 2003 SSDI % TEST 1 TEST 2 TEST 3 MeanMean of Variety SSDI Flower** SSDI Flower SSDI Flower SSDI FlowerConversion* 46A76 03SN40341 5.8 48 7.2 48 7.2 44 6.7 46.7 23 3903SN40441 5.6 46 6.8 49 7.8 45 6.7 46.7 23 39 02SN41269 5.9 44 6.6 456.6 41 6.4 43.3 26 44 46A76 3.8 48 2.0 52 3.4 44 3.1 48.0 59 100 46A652.9 46 3.2 46 3.6 42 3.2 44.7 58 98 44A89 n/a n/a 2.5 47 1.8 46 2.2 46.568 115 *Table 4 conversion 1-9 for SSDI into SSDI % **50% of flower

TABLE 10b Extreme disease pressure field research data on doubledhaploid lines and their parental sources (02SN41269, 03SN40441 and03SN40341) selected for a high level of field resistance. SSDI FLOWERVARIETY PEDIGREE 6.8 48 04DHS11319 POPTC8-03SN40041 Doubled haploid 6.648 04DHS11418 POPTC8-03SN40050 Doubled haploid 7.0 47 04DHS1292103SN40919 Doubled haploid 7.3 47 04DHS12927 03SN40919 Doubled haploid5.8 48 03SN40341 POPTC8-03SN40041 Cycle 8 5.6 46 03SN40441POPTC8-03SN40050 Cycle 8 5.9 44 02SN41269 02SN40102 1.9 49 NS3181BRSusceptible Roundup DH 3.8 48 46A76 Moderately susceptible check 2.9 4646A65 Susceptible check

TABLE 10c Performance of F3 winter canola lines against Sclerotinia(Tavistock, Ontario) and their agronomic/quality traits (Soest, Germany)2005. % mean of combined VARIETY-F3 PEDIGREE SSDIS Conversion checksLodging* Height* Erucic acid 22:1 **Glucosinolates VARIETY-F204CWB930128 03CWB925237-10 6.3 27 42 6.0 7 0.03 19.1 03CWB92523704CWB930127 03CWB925237-7 6.3 27 42 6.0 7.0 0.16 7.9 03CWB92523704CWB930081 03CWB925200-4 5.9 31 48 6.0 4.0 0.03 8.1 03CWB92520004CWB930111 03CWB925024-9 5.8 32 49 6.0 5 0.02 9.9 03CWB92502404CWB930144 03CWB925261-5 5.4 36 55 6.0 3.0 0.10 8.3 03CWB92526104CWB930015 03CWB925059-1 5.3 37 57 8.0 5.0 0.02 18.5 03CWB92505904CWB930135 03CWB925245-3 5.3 37 57 6.0 8.0 0.20 17.2 03CWB925245Columbus Columbus 2.0 70 108 Columbus Express Express 3.0 60 92 6.2 5.10.02 15.0 Express *Lodging (1 lodged - 9 erect) and Height (1 short - 9tall) scores from Soest, Germany, 2005 **Glucosinolate contentdetermined on F3 samples from Soest, Germany, 2005

TABLE 11a Pedigrees of three spring canola selections and their doubledhaploid progenies *Field testing Line # Pedigree Status samples 41269and DHS 04DHS12921 03SN40919 Doubled haploid deposited as ATCC AccessionNo: PTA-6781 04DHS12927 03SN40919 Doubled haploid 03SN40919 02SN40102 F5bulk increase 02SN41269 03SN40918 02SN40102 F4 bulk increase out of02SN41269 2003 onward Deposited as 02SN41269ATCC Accession No: PTA-677702SN41269 02SN40102 F3 from a single F2 plant 2002 02SN40102 01SN41722F2 568 F3s selected for field test 01SN41722 01SN41702 × 01SN41209 F101SN41702 01SN41338 × 01SN41277 F1 01SN41338 POPTC5 × (PGR8493 × (POPTC3× NS1602)) F3 selection out of 60 field-tested 01SN41277 POPTC5 ×(PGR8492 × (POPTC3 × NS2082)) F3 selection out of 33 field-tested01SN41209 POPTC5 × (PGR8488 × (POPTC3 × NS1602)) F3 selection out of 73field-tested 40341 and DHS 04DHS11319 POPTC8-03SN40041 Doubled haploiddeposited as ATCC Accession No: PTA-6780 04SN441521 POPTC8-03SN4004104SN441521-S3 bulk increase out of 04SN40006 2004 onward deposited as03SN40341 ATCC Accession No: PTA-6776 04SN40006 POPTC8-03SN40041 S2 bulkGH increase out of 03SN40341 03SN40341 POPTC8-03SN40041 03SN40341-S1 outfrom single S0 plant 2003 40441 and DHS 04DHS11418 POPTC8-03SN40050Doubled haploid deposited as ATCC Accession No: PTA-6778 04SN441522POPTC8-03SN40050 04SN441522-S3 bulk increase out of 04SN40009 2004onward deposited as 03SN40441 ATCC Accession No: PTA-6779 04SN40009POPTC8-03SN40050 S2 bulk GH increase out of 03SN40441 03SN40441POPTC8-03SN40050 03SN40441-S1 from single S0 plant 2003 41433 and DHS05DHS12897 POPTC9-04SN40049 Doubled haploid; NCIMB Accession No: 4139105SN-41433 POPTC9-04SN40049 S2 bulk increase, deposited as 04SN-414332005 onward NCIMB Accession No: 41389 04SN-41433 POPTC9-04SN40049 S1 outof single S0 plant Cycle 9 2004 41415 and DHS 04DHS12879POPTC9-04SN40047 Doubled haploid; NCIMB Accession No.: 41390 05SN-41415POPTC9-04SN40047 S2 bulk increase, deposited as 04SN-41415 2005 onwardNCIMB Accession No: 41388 04SN-41415 POPTC9-04SN40047 S1 out of singleS0 plant Cycle 9 2004 *segregating material originated from a single S1or F3 plant and was increased for subsequent years of testing after thefirst year

TABLE 11b Pedigrees of 7 winter canola submissions F2** Selected F2 withNCIMB F4**** F3*** a designated Deposit Deposits From a single F2 plantsingle plant out of it 41395 05CWB940127 04CWB930127 03CWB925237-7 4139605CWB940128 04CWB930128 03CWB925237-10 41393 05CWB940081 04CWB93008103CWB925200-4 41394 05CWB940111 04CWB930111 03CWB925024-9 4139805CWB940144 04CWB930144 03CWB925261-5 41397 05CWB940135 04CWB93013503CWB925245-3 41392 05CWB940015 04CWB930015 03CWB925059-1 *F1 progeny ofthe complex cross {[CV0242 × 01CWB940022] × [(00FWB940100 × 00FWB940919)× 01CWB910012]} were selfed and selected in the greenhouse forSclerotinia reaction. **Selection for Sclerotinia reaction and qualitywas made among 400 F2 lines in an unreplicated field screen.***Selection for Sclerotinia reaction, lodging, and quality was madeamong 200 F3 lines in a field test with three replications. Each F3 lineis descended from a single F2 plant. ***F4 bulk increase in plots, Soest2005 harvest for a patent deposit as the increase of the F3 selectedgeneration from a single plant

TABLE 11c Components of complex cross for Sclerotinia resistance inwinter canola. Resistance Component Parentage*** Components GenerationComments 1 01CWB940022 97CWN39131 × (CV0058 × CV0057) PI469955 F4 Indoorscreen (4 out of 16 plants selected) before further crossing 01CWB93002297CWN39131 × (CV0058 × CV0057) PI469955 F3 Indoor screen (4 out of 16plants selected) 01CWB920022 97CWN39131 × (CV0058 × CV0057) PI469955 F2Indoor screen (4 out of 16 plants selected) 01CWB910022 (97CWN39131 ×(CV0058 × CV0057) PI469955 F1 Cross 97CWN39131 CV0065 × PI469955PI469955 F3 selection for canola quality (erucic acid) and resistance 4plants per selection (glucosinolates on seeds)-testing 20 low erucic F2families 96CWN29131 CV0065 × PI469955 PI469955 F2 self to produce 1,300seeds for erucic acid 95CWN19131 CV0065 × PI469955 PI469955 F1 cotyledontest cross 2* 99FWB940100 (CV0043 × CV0063) × 97CWN39191 PI469830 F497CWN39191 CV0114 × PI469830 PI469830 F3 3* 00FWB940919 ((CV0063 ×CV0059) × PI470079 F4 (CV0060 × CV0011)) × 97CWN39260 97CWN39260 CV0087× PI470079 PI470079 F3 4* 01CWB910012 NW3978 × (CV0060 × 00SN42757) PopTCycle 6 BC1F1 Table 8 00SN42757 PopT Cycle 6 (Table PopT Cycle 6 F2 bulkpollen from Cycle 6 Pop T 3,000 plants description 8) (Table 8)**Component 2 and 3 generated in the same way as Component 1 ***CV andNW codes refer to Sclerotinia-susceptible elite varieties from Pioneer'swinter canola collection

Example 3 Canola Determination

According to the Canola Council of Canada, canola is defined by thefollowing properties: The oil must contain less than 2% erucic acid andthe solid component of the seed must contain less than 30 micromoles ofany one or any mixture of 3-butenyl glucosinolate, 4-pentenylglucosinolate, 2-hydroxy-3 butenyl glucosinolate and2-hydroxy-4-pentenyl glucosinolate per gram of air-dry, oil free solid.

The erucic acid level and glucosinolates content were measured to verifythat the seed produced by Population T conforms to the definition ofcanola. The erucic acid level was measured by whole seed fatty acidprofile and the glucosinolate level was measured by scanning NIR asdescribed below:

Fatty Acid Content: The typical percentages by weight of fatty acidspresent in the endogenously formed oil of the mature whole dried seedsare determined. During such determination the seeds are crushed and areextracted as fatty acid methyl esters following reaction with methanoland sodium methoxide. Next the resulting ester is analyzed for fattyacid content by gas liquid chromatography using a capillary column whichallows separation on the basis of the degree of unsaturation and fattyacid chain length. This procedure is described in the work of J. K.Daun, et al., (1983) which is herein incorporated by reference.

Glucosinolate Content. The total glucosinolates of seed at 8.5% moistureas measured by AOCS Official Method AK-1-92 (Determination ofglucosinolates content in rapeseed -colza by HPLC) is expressedmicromoles per gram. Capillary gas chromatography of the trimethylsitylderivatives of extracted and purified desulfoglucosinolates withoptimization to obtain optimum indole glucosinolate detection isdescribed in “Procedures of the Western Canada Canola/RapeseedRecommending Committee Incorporated for the Evaluation andRecommendation for Registration of Canola/Rapeseed Candidate Cultivarsin Western Canada.”

Canola must also meet the requirements of the growing seasonagronomically. For spring canola, the average number of days to reach50% flowering typically falls within the range of 30-90 days (Table 1).In order to control for the growth conditions in any one year or in anyone field, the number of days to flowering is compared with officialcheck varieties growing in the same field and under the same conditions.Table 12 summarizes the results of the glucosinolate, erucic acid, daysto 50% flowering and days to maturity tests in comparison to theofficial WCC/RRC (Western Canada Canola/Rapeseed Recommending CommitteeIncorporated for the Evaluation and Recommendation for Registration ofCanola/Rapeseed Candidate Cultivars in Western Canada) check varieties,46A65 and Q2. As can be seen in Table 12, the plants produced byPopulation T are comparable to the checks and meet the definition forspring canola. Variation within an acceptable range may occur due toenvironmental differences.

TABLE 12 Canola quality/spring habit - erucic acid(C22:1)/glucosinolates/flowering/maturity Variety C22:1*Glucosinolates** Flower 50% Maturity Source Ontario 2004 02SN40209 0.06low 11.98 low 48.3 102.5 Pop T 02SN40680 0.06 low 27.79 low 48.2 102.5Pop T 02SN41269 0.00 low 14.50 low 44.7 100.8 related to Pop T 03SN403410.08 low 11.53 low 47.8 102.7 Pop T 03SN40441 0.13 low 11.42 low 46.2101.3 Pop T 03SN40698 0.10 low 13.59 low 45.0 103.2 Pop T 46A76 0.13 low11.15 low 50.2 102.8 Check 46A65*** 0.11 low 16.24 low 47.0 102.0Official Check Q2*** 0.07 low 13.02 low 50.0 102.0 Official Check Chile04/05 C:22:1 Variety 04DHS11319 0.01 low 15.97 low 59.0 119 03SN4034104DHS11418 0.01 low 12.56 low 63.5 118.5 03SN40441 04DHS12921 0.01 low20.12 low 57.0 114 02SN41269 04DHS12927 0.02 low 20.65 low 57.0 11602SN41269 04SN41415 0.02 low 10.39 low 60 113 Pop T 04SN41433 0.02 low10.38 low 63 116 Pop T 46A65 0.01 low 14.65 low 59.0 114 Official checkChile 05/06 Variety 05DHS12879 0.02 low 9.91 low 62 114 04SN4141505DHS12897 0.02 low 9.98 low 65 114 04SN41433 46A65 0.03 low 14.31 low67 118 Official check *percentage of total fatty acids - Erucic (C22:1)**glucosinolates (u mole - total aliphatic glucs/g airdryed meal)***official registration quality checks

Example 4 Trait Complexity

Table 13 shows the complexity of the genetic segregation in crosses withsusceptible elite material aimed at product development. While theefficacy data in FIG. 1 shows trait performance, the segregation data inTable 13 shows low recovery of partially resistant lines. This indicatesthat the pyramided genetic components result in complex segregation. Itis estimated that three or four genes are conferring partial resistancein these materials. Introgression of these three or four genes intoelite material requires significant effort. The greater the contributionof susceptible elite material, the more difficult the introgression ofthe Sclerotinia resistance genes. For example, it will be easier tointrogress the Sclerotinia resistance genes into material that contains50% susceptible elite material compared to material that contains 75%susceptible elite material. Haploid techniques can be used to fix thesegregating progeny in a similar fashion as was used to fix the sourcesof resistance shown in Tables 10a, 10b and 10c.

TABLE 13 Outcome of breeding activities - recovery of resistantprogenies after crossing partially resistant selections with susceptibleelite lines in 2003 and 2004* # of GH Success plants selections ofSuccess of % Susceptible started in Final # of breeding % breeding %Field parent in GH Field field selected GH- Field- Year Gen Contributiontest test lines started started 2003 BC1F₃ 75 1870 170 3 0.2 1.8 2003 F₂50 500 260 6 1.3 2.3 2004 BC1F₃ 75 2400 455 46 2 11   2004 F₂ 50 122 12220 15 15**  2004 BC1F₃ 25 630 235 39 6 16   *Different sources used indifferent years/projects **Lateness inflated the number of selectionssignificantly. The lines flowered later compared to checks

Example 5 Screening for Blackleg Resistance

Blackleg (Leptosphaeria maculans and other Leptosphaeria species), alsoknown as phoma stem canker, is an internationally important disease ofBrassica, causing significant economic losses in Europe, Australia, andNorth America. (Fitt, et al., 2006) Progenies of Population T werescreened for blackleg resistance by the methods outlined in proceduresof WCC/RRC, in “Procedures of the Western Canada Canola/RapeseedRecommending Committee Incorporated for the Evaluation andRecommendation for Registration of Canola/Rapeseed Candidate Cultivarsin Western Canada”, herein incorporated by reference.

Table 14 describes blackleg ratings from two locations in Western Canadawith the most virulent races of the disease. The selections 02SN41269and 02SN40441 were found to have high levels of adult plant resistanceto blackleg with a 2004 mean rating of 8.7 compared to the susceptiblecheck, Westar, which had a rating of 5.8. (Table 14) Data collected in2005 also indicated that blackleg resistance of 02SN41269 and 03SN40441is superior to 46A76.

Field observations in Tavistock (2004 and 2005) indicate that resistanceof winter lines to blackleg is similar to that of Columbus/Express.

TABLE 14 Reaction of experimental materials to blackleg in WesternCanada, 2004/2005 Plum Killam, Blackleg Coulee, Killam, 2004 AlbertaVariety reaction Manitoba Alberta Mean* 2005 02SN41269 8.9 8.5 8.7 6.303SN40441 8.5 8.8 8.7 5.5 46A65 Resistant 7.0 8.4 7.7 4.6 Check Q2Resistant 7.1 8.0 7.6 N/A Check 03SN40341 7.0 7.5 7.3 4.7 03SN40698 6.08.4 7.2 5.2 02SN40209 6.3 7.8 7.0 N/A 02SN40680 6.3 7.3 6.8 5.2 46A764.9 8.5 6.7 3.6 04DHS11319 3.8 04DHS12921 5.6 04DHS11418 6.5 04SN-414155.8 04SN-41433 5.3 WESTAR Susceptible 6.4 5.2 5.8 3.3 check *1 = deadplant, 9 = no symptoms of disease

Example 6 Greenhouse and Growth Chamber Screening for SclerotiniaResistance

Development of methodologies to screen for Sclerotinia in thegreenhouse/growth rooms was one of the critical success factors indeveloping Sclerotinia resistant Brassica lines. It is well establishedthat generating reliable data for Sclerotinia screening is problematic.

The following method was developed to evaluate canola stem and/or leafreaction to Sclerotinia stem rot in the greenhouse and growth room. Thismethod was used to develop and screen the Sclerotinia resistant lines ofExamples 1, 2, 3, 4 and 5. Although the methods described are directedto Brassica, it is to be understood that any plant susceptible toSclerotinia can be used, for example soybean or sunflower.

Uniformity

Sclerotinia interacts with both the environment and the plant, anddisease development reflects all aspects of that interaction. In orderto obtain the most accurate results in breeding for Sclerotiniaresistance, the maximum uniformity of (1) plant materials (growth stage,stem or leaf size, inoculation point), (2) inoculum, and (3) theenvironment (humidity chambers, growth rooms or compartments) must beattained. This is a requirement for collection of reliable data.

Sclerotinia and Media

PDA (potato dextrose agar) can be used for propagating Sclerotinia. Themedium is rich in nutrients, allowing rapid fungal growth. Also, planttissue can be infected via PDA. Thus, PDA is very good for the initialtransfer, and for situations where higher or faster infection pressureneeds to be exerted, for example in very late growth stages or when ashort turnaround time is required for the material.

For more sensitive tests, PDA low-nutrient should be used. Mycelialgrowth and the infection process are slowed, and the plant materialstands a better chance of expressing its typical reaction. Also, theinfection process can be more reliably interrupted so selections have abetter chance of producing seeds. The steps listed below may befollowed:

Media PDA-Potato Dextrose Agar Media Ingredients 1 L PDA (PotatoDextrose Agar) 39 g. PDA low nutrient Media Ingredients 1 L PDB (PotatoDextrose Broth) 12 g Agar (Sigma A-1296) 15 g

-   1. Retrieve sclerotia of S. sclerotiorum isolate.-   2. Cut sclerotia in half aseptically and place cut side down on a    plate of PDA.-   3. Incubate in the dark at 19° C. (+/−3° C.) for 72 to 90 hours or    until the mycelium nearly reaches the edge of the plate. One can use    lower temperatures to slow down the growth (4-16° C.).-   4. For inoculation in the greenhouse or growth room cut plugs of    approximately 2-4 mm in diameter using a cork borer. It is preferred    to use plugs from the outside ring of mycelium, where mycelium is    uniformly developed, so all plugs are of a similar age and quality.

Stem Inoculation Method

One can inoculate canola after stem elongation until physiologicalmaturity, i.e. seed color change. In the field, the plant is affectedduring the petal drop stage. Generally, early-infected plants are moresusceptible and accordingly older plants are less susceptible. Dependingon the objective of the screening, one can inoculate from pre-floweringto post-flowering. Plants can be grown in 32-cell flats or 4-inch (andbigger) pots. Bigger and more robust plants are more resistant toSclerotinia. Smaller and thinner plants are more susceptible. It is notrecommended to test internodes, but points close to the nodes just abovethe leaf axils, to simulate natural infection. Sclerotinia susceptibleand/or partially resistant checks should be included in the screening.

Entomological Needle Method

For plants tested in a humidity chamber, a needle inoculation, forexample an entomological needle, method can be used. A 3 mm plug (+/−1mm) is prepared and probed with a needle, for example stainlessentomological needle #1 to #3. The needle is used to support theattachment of the plug supporting the mycelium to the stem. The methodenables one to test a large number of plants. The plants may be thin,for example when grown in 32-cell flats. The steps listed below may befollowed:

-   1. Inoculation can be performed at a single or multiple points on    the stem depending on the purpose of the experiment.-   2. Place the plants in the humidity chamber or humid environment for    approximately 20 to 60 hours or until symptoms start developing.    Observe the reaction of the susceptible and/or partially resistant    checks. Remove inoculum (limited term inoculation) and cease the    incubation in humidity when the fresh lesion length on most of the    plants of a susceptible and/or partially resistant check is at least    5-30 mm, depending on the objective of screening. Lesion length is    the most reliable parameter. Initial selections can be made    immediately after incubation in the chamber, as initial reaction is    a good indication of performance.-   3. Move plants to the greenhouse/growth room bench and record the    lesion length and growth stage after incubation where needed. Ensure    that plants are not allowed to dry out. Growth stage can be recorded    prior to inoculation if needed.-   4. Selection should be performed relative to the desired target and    checks or relative to performance of adjacent plants.-   5. If needed for experimental purposes, measure lesion length and    the rate of disease severity (1-9) when satisfactory differentiation    is attained or after one and two weeks. This can vary relative to    the progress of disease development on plants. While rating disease    severity, take into account lesion length, stem stiffness and extent    of girdling.-   6. Avoid conducting inoculation experiments during summer months    when greenhouse temperatures are elevated. If needed, experiments    can be conducted in the growth room, preferably with an increase in    air humidity.

Use of the entomological needles and small-diameter low-nutrient PDAplugs with mycelium has enabled the screening of a large number ofplants and has enabled multiple inoculations on a single plant in orderto verify the reaction. This is very important in selecting andadvancing the most resistant progenies to the field for furtherevaluation.

Sclerotinia rating scale (SSDS indoor—Sclerotinia sclerotiorum diseaseseverity indoor)

1—Prematurely ripened or dead plant

3—Large lesion, weak and completely-girdled stem

5—Large lesion >30 mm, stiff and nearly-girdled stem

7—Small lesion <30 mm, stiff and not-girdled stem

9—No lesion

Intermediate scores can be assigned if symptom severity falls in betweendefined scores.

A less humid and a well aerated environment can be used after infectionto help infected flowering plants survive infection.

Leaf Inoculation Method

Leaf inoculation is performed in order to detect differences in thelevel of resistance of different entries. It is performed at earliergrowth stages than stem inoculation, e.g. pre-bolting, for an earlydetection of partial resistance. However, leaf screening is normallyconducted at flowering, and can occur in the field within whole plantevaluation. As leaves are more sensitive to Sclerotinia compared tostem, low-nutrient PDA should be used unless otherwise specified. Intactleaves can be inoculated with plugs, or plugs with entomologicalneedles, for testing and selection purposes. The steps listed below maybe followed:

-   1. Large-scale screening can be conducted in a humidity chamber. It    is important that the level of moisture in the chamber is    sufficiently high to enable infection to occur, yet not excessive as    free water impedes fungal infection.-   2. Take 2-4 mm plug preferably from the outer 1 cm of colony edge    with uniform mycelial cover and place the plug upside down on the    leaf. Position the plug to leave as much leaf area as possible for    lesion development.-   3. Do not attempt to measure lesion diameter unless there is uniform    development of lesions around the plugs. Avoid having plugs without    good leaf contact. If these occur, count them as escapes. Measure    leaf lesion diameter in millimeters before the fungus has reached    the end of the available leaf tissue on susceptible check or when    deemed appropriate.-   4. Remove the material from the humidity chamber after uniform    lesion establishment around plugs (or good differentiation between    susceptible and resistant checks) and keep material under the    regular humidity conditions. Visual selection relative to the checks    or the adjacent plants is done for both parameters, against    sensitive reaction(s) or combinations of moderate and sensitive    reactions.

Example 7 Field Screening for Sclerotinia Resistance Under ExtremeDisease Pressure Field Research Conditions

Methodology improvements were critical to success in developingSclerotinia-resistant Brassica lines. It is well established thatgenerating reliable field data on an annual basis is not common.Sclerotinia is a potent disease but it only develops during wet summerswith moderate temperatures. A number of issues become critical inscreening for Sclerotinia resistance in the field in years when theconditions of Sclerotinia are sub-optimal. Duration of wetness, waterquality, availability of inoculum, and presence of moist or humidmicroenvironments affect disease development in the crop. Although themethods described are directed to Brassica, it is to be understood thatthe methods may be applied to any plant susceptible to Sclerotiniainfection via ascospores. This includes sunflower (head rot), safflower(head rot), dry bean (pod rot), dry pea (pod rot), soybean (stem and podrot), alfalfa (blossom blight), and lettuce (lettuce drop). Bardin andHuang, 2001. See also, US Patent Application Publication Number2003/0150016 for Sclerotinia effects in soybean.

The critical issues in the field have been resolved as follows:

-   (a) Appropriate artificial inoculum for continuum of data    collection: Since natural inoculum is not always triggered in the    field, an inoculum that mimics infection via petals has been    developed. The carrier for the fungus can be Niger seed (Guizotia    abyssinica-Nyer seed) colonized with Sclerotinia and distributed at    the time of full petal drop.-   (b) Water quality and Sclerotinia: Initially, ground water was used    to irrigate the Sclerotinia colonized fields. However, a lack of    infection transfer in years with low rainfall and either high or low    temperatures was observed. In vitro tests have confirmed that ground    water inhibits Sclerotinia growth. Through lab and field testing, it    was determined that deionizing (DI) water treatment alters the    ground water quality sufficiently to prevent inhibition of    Sclerotinia development. Henceforth, DI water was used to irrigate    extreme disease pressure field research plots. In theory, the    treated deionized water differs from the original ground water in    that the minerals, for example magnesium and calcium (lime), are    eliminated while the pH is not affected. Sclerotinia produces oxalic    acid, a diffusible toxin, to aid in the infection process (U.S. Pat.    No. 6,380,461). Calcium can bind with oxalic acid to create calcium    oxalate. Removal of calcium is very likely the qualitative change in    the deionized water that enables growth of Sclerotinia. Accordingly,    a water source low in minerals or having no minerals, for example    reduced or eliminated magnesium and calcium, can be used.-   (c) Irrigation operated by leaf-wetness sensors: To enable    continuous wetness in the field, leaf wetness sensors (Campbell    Scientific) that trigger irrigation only if moisture is lower than a    set threshold are used. Optimized irrigation enables disease    development and enhances screening for disease resistance. However,    excess irrigation may interfere with meaningful evaluation. In    particular, in a research setting with rows of unique genotypes in    close proximity, lodging of one entry can lead to transfer of the    pathogen by plant-to-plant contact and increased disease incidence    on a second genotype. Thus the Sclerotinia resistance score for the    second genotype may underestimate its potential performance in a    more homogeneous population. In natural field data trials, excessive    irrigation can create a more conducive environment for Sclerotinia    through an increase in lodging over what is usual for a given    genotype. Thus, the performance of the trial entries may be    distorted due to excessive irrigation, such as occurred in the 2005    NDSU test. (See, FIG. 2)-   (d) Providing an enclosure to help maintain a microenvironment    necessary for disease development: To enable development of disease    in dry, hot and/or windy seasons, a netting enclosure may be used.

These new methodologies coupled with the breeding and crossing effortsdescribed above enabled the careful selection of Sclerotinia-resistantcanola lines. The new methodologies enable controlled diseasedevelopment, reliable expression of phenotype, and characterization ofmany different lines under optimal Sclerotinia conditions in order tomake the progress shown in the histogram of FIG. 1.

The following method was developed to screen for Sclerotinia resistancein the field.

Uniformity

Sclerotinia interacts with the environment and plant material. The datagathered reflect_all aspects of this interaction. In order to reducevariability, a maximum uniformity of (1) plant material, (2) inoculumand (3) environment is required. This is a prerequisite for collectionof viable data.

Site Selection/Experimental Design/Planting

-   1. Identify the site that may be colonized with sclerotia for a    natural back-up inoculum together with artificial inoculum. If the    site is not colonized with sclerotia one can attempt to produce    sclerotia via infection of alternative hosts such as soybeans or    white beans, inoculate using a carrier such as Niger seed, or    introduce sclerotia directly.-   2. Plant entries in replicated plots or rows if possible. Use    Randomized Complete Block Design (RCBD) with appropriate susceptible    and/or resistant checks (RCBD has each unit of experimental material    present in each of the blocks (replications)). Attempt to keep    experiments small to decrease the error due to environmental    variation. Running checks should be used and performance expressed    relative to the checks. Maturity of the checks should correspond to    maturity of the lines being tested.-   3. Attempt to grow a dense and healthy canola crop to promote    disease development. Seed rates should be uniform. Precision    planting is preferred, or plants can be thinned to a uniform number    of plants. To promote disease development, consider using a    windbreak by planting strips or additional passes and/or installing    netting around or over the crop.

Favorable Environment/Irrigation/Back-Up Natural Infection

Development of Sclerotinia stem rot is environmentally-dependent and thepresence of inoculum is not sufficient unless a favorable wet or humidmicroenvironment is established within the crop. The relative success ofinfection is measured by the degree to which the susceptible checks areaffected within the experiment.

As natural infection is seldom reliable or uniform, irrigation systemsare used to promote disease development. Irrigation may be initiated assoon as the crop produces an enclosed canopy so that the canopy canretain moisture. Before the onset of flowering, the goal is to keep thetopsoil wet and condition the sclerotia for germination to enable thedevelopment of apothecia in order to produce ascospores for thecolonization of the petals.

Once the petals are colonized, the goal is to enable the progression ofthe disease to the leaves and stems after sufficient petal drop. Thistransfer occurs naturally in years with prolonged wet or humidconditions. Leaf wetness sensors regulate moisture in the canopy bytriggering irrigation when the canopy is dry. The transfer of diseasecan be inhibited with ground water (especially in lower temperatures)so, if feasible, use collected rainwater or deionized ground water.Moisture in the canopy is needed until stem infection on the susceptiblechecks is fully developed.

While the environment is important for disease development, attaineddisease incidence and severity are directly related to the timing of thefollowing factors: favorable environment, growth stage and inoculumpressure.

A netting enclosure can be used to preserve wet or humid conditions andenable disease development.

Inoculum Preparation/Artificial Infection

Artificial inoculum is used as a primary inoculum to increase diseasepressure at the site and enable development of disease similar to theextreme disease pressure. Natural inoculum is a back-up inoculum in thiscase. Niger seeds colonized with Sclerotinia may be used as a carrierfor this purpose. The steps listed below may be followed:

-   1. Prepare PDA plates of Sclerotinia, incubate at 19° C. (+/−3° C.)    for 3-6 days or until the colonies nearly reach the edge of plate as    described in Example 6.-   2. Place five to six mycelial plugs (2-4 mm diameter) into flasks    with 200 ml (+/−100 ml) PDB with 0.5 g/l streptomycin to initiate    production of fresh mycelium.-   3. Incubate in the dark at 19° C. (+/−3° C.) for 2-3 days on the    shaker at 1.2 rpm.-   4. Extract PDB from hyphal mass, homogenize hyphal mass in the    blender, add Tween™ (approximately 0.5 ml/l) and dilute to 1-3 g    (optimum 2 g) of fresh mycelium/1 L of water for field inoculation.    PDB may be added back once the mycelium is weighed out. For    colonizing Niger seed with inoculum use the following procedure:    Autoclave the seed twice using ratio 1:2 of H₂0:Niger. After the    Niger has cooled, add about 100 ml PDB: 500 g Niger and incubate at    room temperature in the dark. One can use straight PDB or dilute it    as needed. Once incubation and the development of the fungal    inoculum on the seed is complete, dry the inoculum and break the    clumps of seeds. This typically takes between 5 and 15 days. To    simulate and enhance natural infection, the inoculum can be applied    during significant petal drop.-   5. Ensure that sufficient Niger inoculum is available before    flowering.-   6. Sclerotinia-colonized Niger seeds are sieved prior to    application. Colonized seeds can be spread over plant material by    hand. For large-scale inoculation, a fertilizer spreader or other    spreading device, for example a mistblower, can be used to    distribute the Niger seeds. The application should be carried out in    the front and the back of each range.-   7. Approximately 5-20 kg/ha of Niger seed are used. The uniformity    and quality of the distribution should be verified.-   8. The goal of the Niger application is to produce a number of leaf    lesions per plant that may progress to stem infection.-   9. The Sclerotinia-colonized Niger seed application should be    repeated if needed.

Rating

The percentage of infected plants and disease severity can be rated(Tables 2 and 15) once the targeted incidence and extent of stemsymptoms have developed on susceptible and/or resistant checks. It isrecommended to conduct one rating when the desired differentiation isattained. Subsequent ratings are less reliable as the results may beaffected by the physical impact on the canopy and stand during theinitial rating. The rating is adjusted relative to the effect that thesymptoms may have on the yield of the affected plant. For example,girdling on the lower stem versus on the higher stem has more effect onyield. Affected side racemes will reduce yield in pods that are only onthat raceme, while an affected main raceme will affect the pods on thewhole plant. This is reflected in the SSDS score; see, Table 15.

Number and position of rated plants per row or plot are to be determinedrelative to the plant and disease development within the experiment.Disease pressure tends to be most uniform in the middle of rows orplots. Atypical and small plants should be excluded from the count andpreferably pulled out of the row prior to or at counting. Thinning earlyin the season would resolve this issue. If possible, outside plantsshould not be rated.

In order to enable viable comparison of material and account forenvironmental variation, trials contain running checks such as 46A65 and46A76.

TABLE 15 Guidelines for scoring severity of symptoms on a single-plantbasis in the field - Pioneer SSDS field - Sclerotinia sclerotiorumdisease severity - field. Numbers in brackets designate public scale.Symptoms Pioneer SSDS Primary Scale of 1 to 9 Branches SecondaryApproximate Public scale 0-5 Main (Off main Branches Yield loss inbrackets( ) Stem stem) (Off primary) Pods (%) 1 (5) Prematurely 50 Ripened Plant - dead 2 (5) Girdled and 40-50 weak - will affect yield 3(4) Girdled but less Most dead 30-40 yield reducing or dying branches 4(3) At least 50% Many dead 20-30 girdled; or dying Some yield branchesimpact 5 (2) Very Few dead 10-20 Large lesion or dying branches 6 (2)Large lesion One dead Multiple dead Multiple  5-10 or dying or dyingInfected branch branches Pods 7 (1) Medium Girdled Few dead or Few 1-5Lesion branch dying infected (~30 mm) Branches pods 8 (1) Small Branchwith One dead or One 1 Lesion non-girdling dying branch infected lesionpod 9 (0) No No No No 0 Symptoms symptoms Symptoms symptoms

The methods of Examples 6 and 7 required significant technical humanintervention and were used in the development of theSclerotinia-resistant lines of the present invention. Significant humantechnical intervention allowed year-round testing of selections andprovided consistent and reproducible results. In addition, the method ofexample 7 allowed the development of extreme disease conditions everyyear, regardless of the natural environment.

Example 8 Producing Inbred Lines Having Resistance to Sclerotinia

Table 13 outlines efforts in inbred line development and success indeveloping elite inbreds in 2003 and 2004. The trait is complex,comprising approximately 3-4 genes in materials from elite crossestested in 2003/2004. Since the material in Population T is canolaquality (Table 12), different approaches as outlined in Table 13 areavailable. Trait recovery (i.e. Sclerotinia resistance) is feasible instraight crosses where elite inbreds provide 50% of the genetics (F₂).

BC1 into resistant sources enables easier recovery of the trait but thecompromise is a lower proportion of the genetic background of the eliteinbreds. It is possible to fully recover resistance by breeding BC2 orhigher with resistant backgrounds if the primary goal is to recoverresistance, with lower consideration for other breeding issues, forexample quality, hybrid vigour, etc. BC1 into susceptible elites isaimed at recovery of elite backgrounds (75%) and the Sclerotiniaresistant trait, but recovery of the Sclerotinia resistant trait becomesmore challenging.

Once elite lines with field resistance are recovered, new breedingapproaches are possible where recovery of resistance becomes morefeasible (field resistant elite crossed by field resistant elite, forexample). Recovering resistance in such crosses is relatively easy andthe F1 material can be subjected to doubled haploidy and resistantprogenies genetically stabilized, i.e. fixed, for a repeatable andpredictable response.

Example 9 Producing Hybrids Having Resistance to Sclerotinia

Due to the fact that partial resistance is not dominant, it is necessaryto have resistance in both the male and female inbreds involved inhybrid seed production. Thus, field resistant inbreds must be developedin female as well in male pools of genetic materials. Hybrid crops areknown for higher yields compared to their inbred components due toheterosis based on the genetic distance between female and malecomponents of a hybrid.

By crossing well-established susceptible female and male inbreds (Table13) with the Sclerotinia-resistant lines of the present invention, elitelines with field resistance are recovered having various combinations ofelite background and resistant background (for example 25-75% of elitebackground as shown in Table 13). Such newly developed female and maleinbreds have both field resistance as well as the genetic distancepreviously established in their elite but susceptible parental lines.Similar to regular hybrid breeding, extensive field testing issubsequently used to determine which inbred combinations provide highyield as well as an adequate level of field resistance to Sclerotinia.

Once a number of elite female and male lines capable of producinghybrids with high yield and field resistance to Sclerotinia areidentified, further progress can be made by crossing such lines anddeveloping inbreds that will further elevate yield as well as fieldresistance to Sclerotinia.

Accordingly, the invention includes not only those lines particularlydescribed herein, but any descendent or progeny, in particular progenyproduced by extracting doubled haploid lines, having the Sclerotiniaresistance trait. The invention also includes any hybrids produced usingthe lines described herein. Further, the invention includes seeds, plantcells and cellular materials, including pollen and ovules, derived fromthe improved plants, lines, or progeny. Plant cells can be isolated fromplants as is known to those skilled in the art. For example, see,Chuong, et al., (1985); Barsby, et al., (Spring 1996); Kartha, et al.,(1974); Narasimhulu, et al., (Spring 1988); and Swanson, (1990).

Plants from the present invention can be used to grow a crop as is knownto those skilled in the art. Further, plants from the present inventioncan be used for oil and meal production. Seeds from plants of thepresent invention can be used to produce canola oil as is known to thoseskilled in the art. The method may include crushing the seeds,extracting crude oil from the seeds, and refining, bleaching anddeodorizing the crude oil to produce the canola oil. Canola meal can beproduced as is known to those skilled in the art. Accordingly, theinvention also includes crushed seeds from the plants of the presentinvention.

Finally, the plants of the present invention can be used for breeding asis known to those skilled in the art, particular examples of which aredescribed below.

V. Further Embodiments of the Invention

The inbred and hybrid lines of examples 8 and 9 can be achieved usingmethods of plant breeding as are known to those skilled in the art, asdescribed above, and as described below. In addition to inbred andhybrid line development, the invention is also directed to methods forusing the Sclerotinia resistant lines of this invention to meet otherplant breeding objectives.

One such embodiment is the method of crossing the Sclerotinia resistantlines of this invention with another canola plant to form a firstgeneration population of F1 plants. The population of first generationF1 plants produced by this method is also an embodiment of theinvention. This first generation population of F1 plants will comprisean essentially complete set of the alleles of the Sclerotinia resistantlines of this invention. Typically in the art an F1 hybrid is consideredto have all the alleles of each homozygous parent. One of ordinary skillin the art can utilize either breeder books or molecular methods toidentify a particular F1 plant produced using the Sclerotinia resistantlines of this invention, and any such individual plant is alsoencompassed by this invention. These embodiments also cover use of thesemethods with transgenic or single gene conversions of the Sclerotiniaresistant lines of this invention.

Another embodiment of this invention is a method of using theSclerotinia resistant lines of this invention in breeding that involvesthe repeated backcrossing to the Sclerotinia resistant lines of thisinvention any number of times. Using backcrossing methods, or thetransgenic methods described herein, the single gene conversion methodsdescribed herein, or other breeding methods known to one of ordinaryskill in the art, one can develop individual plants and populations ofplants that retain at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%,70%, 75%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5% of the geneticprofile of the Sclerotinia resistant lines of this invention. Thepercentage of the genetics retained in the progeny may be measured byeither pedigree analysis or through the use of genetic techniques suchas molecular markers or electrophoresis. In pedigree analysis, onaverage 50% of the starting germplasm would be passed to the progenyline after one cross to another line, 25% after another cross to adifferent line, and so on. Molecular markers could also be used toconfirm and/or determine the pedigree of the progeny line.

A specific method for producing a line derived from the Sclerotiniaresistant lines of this invention is as follows. One of ordinary skillin the art would cross a Sclerotinia resistant line of this inventionwith another canola plant, such as an elite line. The F1 seed derivedfrom this cross would contain a single copy of 100% of the alleles fromthe Sclerotinia resistant line of this invention and a single copy of100% of the alleles of the other plant. The F1 seed would be grown toform a homogeneous population and allowed to self, thereby forming F2seed. On average the F2 seed would have derived 50% of its alleles fromthe Sclerotinia resistant line of this invention and 50% from the othercanola plant, but various individual plants from the population wouldhave a much greater percentage of their alleles derived from theSclerotinia resistant lines of this invention (Wang and Bernardo, 2000and Bernardo and Kahler, 2001). The F2 seed would be grown and selectionof plants would be made based on visual observation and/or measurementof traits. The traits used for selection may be the traits associatedwith the Sclerotinia resistant lines of this invention. The derivedprogeny that exhibit the desired traits of the Sclerotinia resistantlines of this invention would be selected and each plant would beharvested separately. This F3 seed from each plant would be grown inindividual rows and allowed to self. Then selected rows or plants fromthe rows would be harvested and threshed individually. The selectionswould again be based on visual observation and/or measurements fordesirable traits of the plants. The process of growing and selectionwould be repeated any number of times until an inbred of the Sclerotiniaresistant line of this invention is obtained. The inbred of theSclerotinia resistant line of this invention would contain theSclerotinia resistant trait.

If the other canola plant to which the Sclerotinia resistant line wascrossed also contained Sclerotinia resistance genes, then an inbreddeveloped from the progeny may exhibit Sclerotinia resistance at a levelequal to or greater than the level expressed in the Sclerotiniaresistant line of this invention. An inbred would have, on average, 50%of its genes derived from the Sclerotinia resistant lines of thisinvention, but various individual plants from the population would havea much greater percentage of their alleles derived from the Sclerotiniaresistant line of this invention. The breeding process of crossing,selfing, and selection may be repeated to produce another population ofthe Sclerotinia resistant lines of the invention-derived canola plantswith, on average, 25% of their genes derived from the Sclerotiniaresistant line of this invention, but various individual plants from thepopulation would have a much greater percentage of their alleles derivedfrom the Sclerotinia resistant line of this invention.

The previous example can be modified in numerous ways; for instance,selection may or may not occur at every selfing generation, selectionmay occur before or after the actual self-pollination process occurs, orindividual selections may be made by harvesting individual pods, plants,rows, or plots at any point during the breeding process described. Inaddition, doubled haploid breeding methods may be used at any step inthe process. The population of plants produced at each and anygeneration of selfing is also an embodiment of the invention.

Another embodiment of this invention is the method of obtaininghomozygous Sclerotinia resistant lines by crossing a Sclerotiniaresistant line of this invention with another canola plant and applyingdoubled haploid methods to the F1 seed or F1 plant or to any generationof the Sclerotinia resistant lines of this invention obtained by theselfing of this cross.

Still further, this invention also is directed to methods for producingthe Sclerotinia resistant lines of this invention by crossing aSclerotinia resistant line of this invention with a canola plant andgrowing the progeny seed, and repeating the crossing and the growingsteps with the Sclerotinia resistant line of this invention from 1 to 2times, 1 to 3 times, 1 to 4 times, or 1 to 5 times and selfing anynumber of times after the first, second, third, fourth, or fifth cross.

Thus, any and all methods using one or more of the Sclerotinia resistantlines of this invention in breeding are part of this invention,including selfing, pedigree breeding, backcrosses, hybrid production andcrosses to populations. All plants and populations of plants producedusing one or more of the Sclerotinia resistant lines of this inventionas a parent are within the scope of this invention. Unique molecularmarker profiles and/or breeding records can be used by those of ordinaryskill in the art to identify the progeny lines or populations of progenyderived from one or more of the Sclerotinia resistant lines of thisinvention.

All plants produced using a Sclerotinia resistant line of this inventionas a parent are within the scope of this invention, including thosedeveloped from progeny derived from the inbred of a Sclerotiniaresistant line of this invention.

A further embodiment of the invention is a single gene conversion of theSclerotinia resistant lines of this invention. A single gene conversionoccurs when DNA sequences are introduced through traditional(non-transformation) breeding techniques, such as backcrossing. DNAsequences, whether naturally-occurring or transgenes, may be introducedusing these traditional breeding techniques. Desired traits transferredthrough this process include, but are not limited to, fertilitymodification, fatty acid profile modification, other nutritionalenhancements, industrial enhancements, disease resistance, insectresistance, herbicide resistance and yield enhancements. The trait ofinterest is transferred from the donor parent to the recurrent parent,in this case, the canola plant disclosed herein. Single gene traits mayresult from the transfer of either a dominant allele or a recessiveallele. Selection of progeny containing the trait of interest is done bydirect selection for a trait associated with a dominant allele.Selection of progeny for a trait that is transferred via a recessiveallele requires growing and selfing the first backcross to determinewhich plants carry the recessive allele. Recessive traits may requireadditional progeny testing in successive backcross generations todetermine the presence of the gene of interest. Along with selection forthe trait of interest, progeny are selected for the phenotype of therecurrent parent. It should be understood that occasionally additionalpolynucleotide sequences or genes are transferred along with the singlegene conversion trait of interest. Progeny containing at least 90%, 95%,96%, 97%, 98%, 99% or 99.5% of the genes from the recurrent parent, thecanola plant disclosed herein, plus containing the single geneconversion trait, is considered to be a single gene conversion of theSclerotinia resistant lines of this invention.

It should be understood that the Sclerotinia resistant lines of theinvention can, through routine manipulation of cytoplasmic genes,nuclear genes, or other factors, be produced in a male-sterile form asdescribed in the references discussed earlier. Such embodiments are alsowithin the scope of the present claims. The Sclerotinia resistant linesof this invention can be manipulated to be male sterile by any of anumber of methods known in the art, including by the use of mechanicalmethods, chemical methods, self-incompatibility, cytoplasmic malesterility (either ogura or another system) or nuclear male sterility.The term “manipulated to be male sterile” refers to the use of anyavailable techniques to produce a male sterile version of a Sclerotiniaresistant line of this invention. The male sterility may be eitherpartial or complete male sterility. This invention is also directed toF1 hybrid seed and plants produced by the use of the Sclerotiniaresistant lines of this invention.

This invention is also directed to the use of the Sclerotinia resistantlines of this invention in tissue culture. As used herein, the termplant cell includes plant protoplasts, plant cell tissue cultures fromwhich canola plants can be regenerated, plant calli, plant clumps, andplant cells that are intact in plants or parts of plants, such asembryos, pollen, ovules, seeds, flowers, ears, silique, leaves, stems,roots, root tips, anthers, cotyledons and the like. Tissue culture aswell as microspore culture for regeneration of canola plants can beaccomplished successfully. (Chuong, et al., (1985); Barsby, et al.,(Spring 1996); Kartha, et al., (1974); Narasimhulu, et al., (Spring1988); Swanson, (1990). Thus, it is clear from the literature that thestate of the art is such that these methods of obtaining plants are, andwere, “conventional” in the sense that they are routinely used and havea very high rate of success.

The utility of the Sclerotinia resistant lines of this invention alsoextends to crosses with other species. Commonly, suitable species willbe of the family Brassica. In particular, Sclerotinia-resistant winterlines may be sources of resistance in breeding programs for spring orsemi-winter lines. Sclerotinia-resistant spring lines may be sources ofresistance in breeding programs for semi-winter or winter lines. Allsuch uses are contemplated by, and made a part of, the presentinvention.

The advent of new molecular biological techniques have allowed theisolation and characterization of genetic elements with specificfunctions, such as encoding specific protein products. Scientists in thefield of plant biology have developed a strong interest in engineeringthe genome of plants to contain and express foreign genetic elements, oradditional or modified versions of native or endogenous geneticelements, in order to alter the traits of a plant in a specific manner.Any DNA sequences, whether from a different species or from the samespecies that are inserted into the genome of the species usingtransformation are referred to herein collectively as “transgenes”. Theprocess of “transforming” is the insertion of DNA into the genome. Overthe last fifteen to twenty years, several methods for producingtransgenic plants have been developed, and the present invention, inparticular embodiments, also relates to transformed versions of theclaimed Sclerotinia resistant lines of this invention.

Numerous methods for plant transformation have been developed, includingbiological and physical plant transformation protocols. See, forexample, Miki, et al., (1988). In addition, expression vectors and invitro culture methods for plant cell or tissue transformation andregeneration of plants are available. See, for example, Evans, et al.,(1983), Binding (1985) and Weissbach, et al., 1988.

The most prevalent types of plant transformation involve theconstruction of an expression vector. Such a vector comprises a DNAsequence that contains a gene under the control of, or operativelylinked to, a regulatory element, for example a promoter. The vector maycontain one or more genes and one or more regulatory elements.

A genetic trait which has been engineered into a particular canola plantusing transformation techniques could be moved into another line usingtraditional breeding techniques that are well known in the plantbreeding arts. For example, a backcrossing approach could be used tomove a transgene from a transformed canola plant to an elite inbred lineand the resulting progeny would comprise a transgene. Also, if an inbredline was used for the transformation then the transgenic plants could becrossed to a different line in order to produce a transgenic hybridcanola plant. As used herein, “crossing” can refer to a simple X by Ycross, or the process of backcrossing, depending on the context. Variousgenetic elements can be introduced into the plant genome usingtransformation. These elements include but are not limited to genes;coding sequences; inducible, constitutive, and tissue specificpromoters; enhancing sequences; and signal and targeting sequences. See,U.S. Pat. No. 6,222,101 which is herein incorporated by reference.

With transgenic plants according to the present invention, a foreignprotein can be produced in commercial quantities. Thus, techniques forthe selection and propagation of transformed plants, which are wellunderstood in the art, yield a plurality of transgenic plants which areharvested in a conventional manner, and a foreign protein then can beextracted from a tissue of interest or from total biomass. Proteinextraction from plant biomass can be accomplished by known methods whichare discussed, for example, by Heney and Orr (1981).

A genetic map can be generated, primarily via conventional RestrictionFragment Length Polymorphisms (RFLP), Polymerase Chain Reaction (PCR)analysis, and Simple Sequence Repeats (SSR) which identifies theapproximate chromosomal location of the integrated DNA molecule codingfor the foreign protein. For exemplary methodologies in this regard,see, Glick and Thompson, (1993). Map information concerning chromosomallocation is useful for proprietary protection of a subject transgenicplant. If unauthorized propagation is undertaken and crosses made withother germplasm, the map of the integration region can be compared tosimilar maps for suspect plants, to determine if the latter have acommon parentage with the subject plant. Map comparisons could involvehybridizations, RFLP, PCR, SSR and sequencing, all of which areconventional techniques.

Likewise, by means of the present invention, plants can be geneticallyengineered to express various phenotypes of agronomic interest.Exemplary transgenes implicated in this regard include, but are notlimited to, those categorized below.

1. Genes that Confer Resistance to Pests or Disease and that Encode:

(A) Plant disease resistance genes. Plant defenses are often activatedby specific interaction between the product of a disease resistance gene(R) in the plant and the product of a corresponding avirulence (Avr)gene in the pathogen. A plant variety can be transformed with clonedresistance gene to engineer plants that are resistant to specificpathogen strains. See, for example, Jones, et al., (1993); Mindrinos, etal.

(B) A protein conferring resistance to fungal pathogens, such as oxalateoxidase or oxalate decarboxylase (Zhou, et al., (1998), U.S. Pat. Nos.3,303,846 and 6,297,425).

(C) A Bacillus thuringiensis protein, a derivative thereof or asynthetic polypeptide modeled thereon. See, for example, Geiser, et al.,(1986), who disclose the cloning and nucleotide sequence of a Btδ-endotoxin gene. Moreover, DNA molecules encoding δ-endotoxin genes canbe purchased from American Type Culture Collection (Manassas, Va.), forexample, under ATCC Accession Numbers 40098, 67136, 31995 and 31998.

(D) A lectin. See, for example, the disclosure by Van Damme, et al.,(1994), who disclose the nucleotide sequences of several Clivia miniatamannose-binding lectin genes.

(E) A vitamin-binding protein such as avidin. See, PCT ApplicationNumber US93/06487, the contents of which are hereby incorporated byreference. The application teaches the use of avidin and avidinhomologues as larvicides against insect pests.

(F) An enzyme inhibitor, for example, a protease or proteinase inhibitoror an amylase inhibitor. See, for example, Abe, et al., (1987)(nucleotide sequence of rice cysteine proteinase inhibitor), Huub, etal., (1993) (nucleotide sequence of cDNA encoding tobacco proteinaseinhibitor I), Sumitani, et al., (1993) (nucleotide sequence ofStreptomyces nitrosporeus α-amylase inhibitor) and U.S. Pat. No.5,494,813 (Hepher and Atkinson, issued Feb. 27, 1996).

(G) An insect-specific hormone or pheromone such as an ecdysteroid andjuvenile hormone, a variant thereof, a mimetic based thereon, or anantagonist or agonist thereof. See, for example, the disclosure byHammock, et al., (1990), of baculovirus expression of cloned juvenilehormone esterase, an inactivation of juvenile hormone.

(H) An insect-specific peptide or neuropeptide which, upon expression,disrupts the physiology of the affected pest. For example, see thedisclosures of Regan, (1994) (expression cloning yields DNA coding forinsect diuretic hormone receptor), and Pratt, et al., (1989) (anallostatin is identified in Diploptera puntata). See also, U.S. Pat. No.5,266,317 to Tomalski, et al., who disclose genes encodinginsect-specific, paralytic neurotoxins.

(I) An insect-specific venom produced in nature by a snake, a wasp, etc.For example, see Pang, et al., (1992), for disclosure of heterologousexpression in plants of a gene coding for a scorpion insectotoxicpeptide.

(J) An enzyme responsible for a hyperaccumulation of a monterpene, asesquiterpene, a steroid, hydroxamic acid, a phenylpropanoid derivativeor another non-protein molecule with insecticidal activity.

(K) An enzyme involved in the modification, including thepost-translational modification, of a biologically active molecule; forexample, a glycolytic enzyme, a proteolytic enzyme, a lipolytic enzyme,a nuclease, a cyclase, a transaminase, an esterase, a hydrolase, aphosphatase, a kinase, a phosphorylase, a polymerase, an elastase, achitinase and a glucanase, whether natural or synthetic. See, PCTApplication Number WO 93/02197 in the name of Scott, et al., whichdiscloses the nucleotide sequence of a callase gene. DNA molecules whichcontain chitinase-encoding sequences can be obtained, for example, fromthe ATCC under Accession Numbers 39637 and 67152. See also, Kramer, etal., (1993), who teach the nucleotide sequence of a cDNA encodingtobacco hookworm chitinase, and Kawalleck, et al., (1993), who providethe nucleotide sequence of the parsley ubi4-2 polyubiquitin gene.

(L) A molecule that stimulates signal transduction. For example, see thedisclosure by Botella, et al., (1994), of nucleotide sequences for mungbean calmodulin cDNA clones, and Griess, et al., (1994), who provide thenucleotide sequence of a maize calmodulin cDNA clone.

(M) A hydrophobic moment peptide. See PCT Application Number WO95/16776(disclosure of peptide derivatives of Tachyplesin which inhibit fungalplant pathogens) and PCT Application Number WO95/18855 (teachessynthetic antimicrobial peptides that confer disease resistance), therespective contents of which are hereby incorporated by reference.

(N) A membrane permease, a channel former or a channel blocker. Forexample, see the disclosure by Jaynes, et al., (1993), of heterologousexpression of a cecropin-β lytic peptide analog to render transgenictobacco plants resistant to Pseudomonas solanacearum.

(O) A viral-invasive protein or a complex toxin derived therefrom. Forexample, the accumulation of viral coat proteins in transformed plantcells imparts resistance to viral infection and/or disease developmenteffected by the virus from which the coat protein gene is derived, aswell as by related viruses. See, Beachy, et al., (1990). Coatprotein-mediated resistance has been conferred upon transformed plantsagainst alfalfa mosaic virus, cucumber mosaic virus, tobacco streakvirus, potato virus X, potato virus Y, tobacco etch virus, tobaccorattle virus and tobacco mosaic virus.

(P) An insect-specific antibody or an immunotoxin derived therefrom.Thus, an antibody targeted to a critical metabolic function in theinsect gut would inactivate an affected enzyme, killing the insect. Cf.Taylor, et al., (1994) (enzymatic inactivation in transgenic tobacco viaproduction of single-chain antibody fragments).

(Q) A virus-specific antibody. See, for example, Tavladoraki, et al.,(1993), who show that transgenic plants expressing recombinant antibodygenes are protected from virus attack.

(R) A developmental-arrestive protein produced in nature by a pathogenor a parasite. Thus, fungal endo α-1,4-D-polygalacturonases facilitatefungal colonization and plant nutrient release by solubilizing plantcell wall homo-α-1,4-D-galacturonase. See, Lamb, et al., (1992). Thecloning and characterization of a gene which encodes a beanendopolygalacturonase-inhibiting protein is described by Toubart, etal., (1992).

(S) A developmental-arrestive protein produced in nature by a plant. Forexample, Logemann, et al., (1992), have shown that transgenic plantsexpressing the barley ribosome-inactivating gene have an increasedresistance to fungal disease.

(T) Gene products involved in the Systemic Acquired Resistance (SAR)Response and/or the pathogenesis related gene products. Briggs, (1995).

(U) Antifungal gene products (Cornelissen and Melchers, (1993) andParijs, et al., (1991) and Bushnell, et al., (1998)).

2. Genes that Confer Resistance to a Herbicide, for Example:

(A) A herbicide that inhibits the growing point or meristem, such as animidazolinone or a sulfonylurea. Exemplary genes in this category codefor mutant ALS and AHAS enzyme as described, for example, by Lee, etal., (1988), and Miki, et al., (1990), respectively.

(B) Glyphosate (resistance imparted by mutant5-enolpyruvl-3-phosphikimate synthase (EPSP) and aroA genes,respectively) and other phosphono compounds such as glufosinate(phosphinothricin acetyl transferase, PAT) and Streptomyceshygroscopicus phosphinothricin-acetyl transferase, bar, genes), andpyridinoxy or phenoxy propionic acids and cycloshexones (ACCaseinhibitor-encoding genes). See, for example, U.S. Pat. No. 4,940,835 toShah, et al., which discloses the nucleotide sequence of a form of EPSPwhich can confer glyphosate resistance. A DNA molecule encoding a mutantaroA gene can be obtained under ATCC Accession Number 39256, and thenucleotide sequence of the mutant gene is disclosed in U.S. Pat. No.4,769,061 to Comai. European Patent Application Number 0 333 033 toKumada, et al., and U.S. Pat. No. 4,975,374 to Goodman, et al., disclosenucleotide sequences of glutamine synthetase genes which conferresistance to herbicides such as L-phosphinothricin. The nucleotidesequence of a phosphinothricin-acetyl-transferase gene is provided inEuropean Application Number 0 242 246 to Leemans, et al., (1989),describe the production of transgenic plants that express chimeric bargenes coding for phosphinothricin acetyl transferase activity. Exemplaryof genes conferring resistance to phenoxy propionic acids andcycloshexones, such as sethoxydim and haloxyfop, are the Acc1-S1,Acc1-S2 and Acc1-S3 genes described by Marshall, et al., (1992).

(C) A herbicide that inhibits photosynthesis, such as a triazine (psbAand gs+ genes) and a benzonitrile (nitrilase gene). Przibilla, et al.,(1991), describe the transformation of Chlamydomonas with plasmidsencoding mutant psbA genes. Nucleotide sequences for nitrilase genes aredisclosed in U.S. Pat. No. 4,810,648 to Stalker, and DNA moleculescontaining these genes are available under ATCC Accession Numbers 53435,67441 and 67442. Cloning and expression of DNA coding for a glutathioneS-transferase is described by Hayes, et al., (1992).

3. Genes that Confer or Contribute to a Value-Added Trait, Such as:

(A) Modified fatty acid metabolism, for example, by transforming a plantwith an antisense gene of stearoyl-ACP desaturase to increase stearicacid content of the plant. See, Knultzon, et al., (1992).

(B) Decreased phytate content

-   -   (1) Introduction of a phytase-encoding gene would enhance        breakdown of phytate, adding more free phosphate to the        transformed plant. For example, see, Van Hartingsveldt, (1993),        for a disclosure of the nucleotide sequence of an Aspergillus        niger phytase gene.    -   (2) A gene could be introduced that reduces phytate content. In        maize, this could be accomplished, for example, by cloning and        then reintroducing DNA associated with the single allele which        is responsible for maize mutants characterized by low levels of        phytic acid. See, Raboy, et al., (1990).

(C) Modified carbohydrate composition effected, for example, bytransforming plants with a gene coding for an enzyme that alters thebranching pattern of starch. See, Shiroza, et al., (1988) (nucleotidesequence of Streptococcus mutans fructosyltransferase gene), Steinmetz,et al., (1985) (nucleotide sequence of Bacillus subtilis levansucrasegene), Pen, et al., (1992) (production of transgenic plants that expressBacillus licheniformis α-amylase), Elliot, et al., (1993) (nucleotidesequences of tomato invertase genes), Sogaard, et al., (1993)(site-directed mutagenesis of barley α-amylase gene), and Fisher, etal., (1993) (maize endosperm starch branching enzyme II).

(D) Reduced green seed, by down regulation of the CAB gene in Canolaseed (Morisette et al., 1997)

(E) Elevated oleic acid via FAD-2 gene modification and/or decreasedlinolenic acid via FAD-3 gene modification (see, U.S. Pat. Nos.6,063,947; 6,323,392; and WO 93/11245).

4. Genes that Control Pollination or Hybrid Seed Production; forExample, Canadian Patent Number 2,087,703.

INDUSTRIAL APPLICABILITY

The seed of the Sclerotinia resistant lines of this invention, the plantproduced from such seed, the hybrid canola plant produced from thecrossing of the Sclerotinia resistant lines of this invention, theresulting hybrid seed, and various parts of the hybrid canola plant canbe utilized in the production of an edible vegetable oil or other foodproducts in accordance with known techniques. For example, a method ofproducing canola oil may comprise: (a) crushing canola seed; (b)extracting crude oil; and (c) refining, bleaching and deodorizing thecrude oil to produce canola oil. The remaining solid meal componentderived from seeds can be used as a nutritious livestock feed.

VI. Deposits

Certain deposits of seed have been made with the American Type CultureCollection (ATCC), Manassas, Va. 20852, which is the deposits include2500 seeds of each of 02SN41269 (F4), PTA-6777; 04DHS12921 (doubledhaploid), PTA-6781; 03SN40341 (F4), PTA-6776; 04DHS11319 (doubledhaploid), PTA-6780; 03SN40441 (F4), PTA-6779; 04DHS11418 (doubledhaploid), PTA-6778. The seeds deposited with ATCC were and have beenmaintained by Pioneer Hi-Bred International, Inc., 800 Capital Square,400 Locust Street, Des Moines, Iowa 50309-2340, since prior to thefiling date of this application. The deposits will be maintained in theATCC depository, which is a public depository, for a period of 30 years,or 5 years after the most recent request, or for the effective life ofthe patent, whichever is longer, and will be replaced if they becomenonviable during that period. Additionally, Applicant has satisfied allthe requirements of 37 C.F.R. Sections 1.801-1.809. Applicant imposes norestrictions on the availability of the deposited material from theATCC; however, Applicant has no authority to waive any restrictionsimposed by law on the transfer of biological material or itstransportation in commerce. Applicant does not waive any infringement ofhis rights granted under this patent. Access to these deposits will beavailable during the pendency of the application to the Commissioner ofPatents and Trademarks and to persons determined by the Commissioner tobe entitled thereto upon request. Applicant does not waive anyinfringement of his rights granted in any under this patent and/or underthe Plant Variety Protection Act (7 USC 2321 et seq.).

Additional deposits of seed were made in May 2006 with NCIMB Ltd., ofAberdeen, Scotland, a public depository recognized by the BudapestTreaty, as follows:

NCIMB 41388 Brassica napus 04SN 41415NCIMB 41389 Brassica napus 04SN 41433NCIMB 41390 Brassica napus 05DHS 12879NCIMB 41391 Brassica napus 05DHS 12897Deposit date 12 May 2006.NCIMB 41392 Brassica napus 04CWB 930015NCIMB 41393 Brassica napus 04CWB 930081NCIMB 41394 Brassica napus 04CWB 930111NCIMB 41395 Brassica napus 04CWB 930127NCIMB 41396 Brassica napus 04CWB 930128NCIMB 41397 Brassica napus 04CWB 930135NCIMB 41398 Brassica napus 04CWB 930144Deposit date 15 May 2006

Details regarding these deposited lines are shown in Tables 11a and 11b.In each case, 3000 seed were deposited, except for NCIMB 41393, forwhich only 400 seeds were deposited due to limited supply. Asupplemental deposit can be made, if needed.

Additionally, a deposit of isolate SS#4 of Sclerotinia sclerotiorum wasmade to the International Depositary Authority of Canada, Winnipeg,Manitoba on May 17, 2006, and assigned accession number 170506-01. Thisisolate was used for all indoor screening and selection methods, andextreme disease pressure field research conditions, described herein.Natural field research data, such as is set forth in Tables 9a, 9b and9c, reflects testing against the natural population of Sclerotinia andconfirms the efficacy of breeding efforts using the SS#4 isolate.

The foregoing invention has been described in detail by way ofillustration and example for purposes of exemplification. However, itwill be apparent that changes and modifications such as single genemodifications and mutations, seasonal variants, variant individualsselected from populations of the plants of the lines described, and thelike, are considered to be within the scope of the present invention.All references disclosed herein, whether to journal, patents, publishedapplications or the like, are hereby incorporated in their entirety byreference.

It should be understood that certain modifications should be and will beapparent to those of ordinary skill in the art, and that suchmodifications to the precise lines, varieties, and procedures describedin the invention are intended to come within the scope of the invention.

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1. Brassica line 03SN40341, representative seed of said line having beendeposited as ATCC Accession Number PTA-6776.
 2. A seed of the brassicaline of claim
 1. 3. A plant of the brassica line of claim
 1. 4. A plantcell from the plant of claim
 3. 5. Progeny of the plant of claim 3,wherein said progeny are representative of a population having an SSDI %score which is less than about 60% of the SSDI % score of PioneerHi-Bred variety 46A76 or of Pioneer Hi-Bred 46A65 or of the mean scoreof said two varieties, and further wherein said progeny produces seedhaving a glucosinolate level of less than 30 μmoles per gram of oil-freesolid and less than 2% erucic acid in the endogenous oil component. 6.The progeny of claim 5 wherein said progeny originate by culture ofhaploid tissue.
 7. The progeny of claim 6 wherein said progeny arerepresented by seed deposited as ATCC accession number PTA-6780.
 8. Amethod of generating progeny of the plant of claim 3, comprising: (a)crossing said plant of claim 3 with itself or with another Brassicaplant to yield seed; (b) growing the Brassica seed of step (a) to yielda derived plant; (c) optionally repeating the crossing and growing ofsteps (a) and (b) for successive generations to produce further plantsderived from the Brassica plant of claim
 3. 9. The method of claim 8,further comprising the step of (d) selecting a descendent plantrepresentative of a population having an SSDI % score which is less thanabout 60% of the SSDI % score of Pioneer Hi-Bred variety 46A76 or ofPioneer Hi-Bred 46A65 or the mean score of said two varieties.
 10. Aplant or group of plants produced by the method of claim
 8. 11. Use ofthe plants of claim 10 for growing a crop.
 12. Use of the plant of claim10 for oil or meal production.
 13. A method of producing an oil,comprising: a. crushing seeds produced by the plant of claim 10; and b.extracting a crude oil from such crushed seeds.
 14. The plants of claim10 further representing a population having an average level of blacklegresistance which is greater than that of Pioneer Hi-Bred variety 46A76under the same environmental and disease conditions.
 15. The plant ofclaim 10 further comprising a transgene.
 16. The plant of claim 10wherein said plant is male sterile.